Markers of acute myeloid leukemia stem cells

ABSTRACT

Markers of acute myeloid leukemia stem cells (AMLSC) are identified. The markers are differentially expressed in comparison with normal counterpart cells, and are useful as diagnostic and therapeutic targets.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 16/738,913, filed Jan. 9, 2020, which is a Continuation in partof application Ser. No. 15/704,790 filed Sep. 14, 2017, now U.S. Pat.No. 10,662,242 issued May 26, 2020, which is a Continuation ofapplication Ser. No. 14/927,349 filed Oct. 29, 2015, now U.S. Pat. No.9,796,781 issued Oct. 24, 2017, which is a Continuation of applicationSer. No. 14/164,009 filed Jan. 24, 2014, now U.S. Pat. No. 9,193,955issued Nov. 24, 2015, which is a Continuation of application Ser. No.13/739,788 filed Jan. 11, 2013, now U.S. Pat. No. 8,709,429 issued Apr.29, 2014, which is a Continuation of application Ser. No. 12/836,152filed Jul. 14, 2010, now U.S. Pat. No. 8,361,736 issued Jan. 29, 2013,which is a Continuation in Part and claims the benefit of PCTApplication No. PCT/US2009/000224, filed Jan. 13, 2009, which claimsbenefit of U.S. Provisional Patent Application No. 61/011,324, filedJan. 15, 2008, which applications are incorporated herein by referencein their entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract CA086017awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Basic cancer research has focused on identifying the genetic changesthat lead to cancer. This has led to major advances in our understandingof the molecular and biochemical pathways that are involved intumorigenesis and malignant transformation. However, our understandingof the cellular biology has lagged. Although the effects of particularmutations on the proliferation and survival of model cells, such asfibroblasts or cell lines, can be predicted, the effects of suchmutations on the actual cells involved in specific cancers is largelyguesswork.

A tumor can be viewed as an aberrant organ initiated by a tumorigeniccancer cell that acquired the capacity for indefinite proliferationthrough accumulated mutations. In this view of a tumor as an abnormalorgan, the principles of normal stem cell biology can be applied tobetter understand how tumors develop. Many observations suggest thatanalogies between normal stem cells and tumorigenic cells areappropriate. Both normal stem cells and tumorigenic cells have extensiveproliferative potential and the ability to give rise to new (normal orabnormal) tissues. Both tumors and normal tissues are composed ofheterogeneous combinations of cells, with different phenotypiccharacteristics and different proliferative potentials.

Because most tumors have a clonal origin, the original tumorigeniccancer cell gives rise to phenotypically diverse progeny, includingcancer cells with indefinite proliferative potential, as well as cancercells with limited or no proliferative potential. This suggests thattumorigenic cancer cells undergo processes that are analogous to theself-renewal and differentiation of normal stem cells. Tumorigenic cellscan be thought of as cancer stem cells that undergo an aberrant andpoorly regulated process of organogenesis analogous to what normal stemcells do. Although some of the heterogeneity in tumors arises as aresult of continuing mutagenesis, it is likely that heterogeneity alsoarises through the aberrant differentiation of cancer cells.

It is well documented that many types of tumors contain cancer cellswith heterogeneous phenotypes, reflecting aspects of the differentiationthat normally occurs in the tissues from which the tumors arise. Thevariable expression of normal differentiation markers by cancer cells ina tumor suggests that some of the heterogeneity in tumors arises as aresult of the anomalous differentiation of tumor cells. Examples of thisinclude the variable expression of myeloid markers in chronic myeloidleukaemia, the variable expression of neuronal markers within peripheralneuroectodermal tumors, and the variable expression of milk proteins orthe estrogen receptor within breast cancer.

It was first extensively documented for leukemia and multiple myelomathat only a small subset of cancer cells is capable of extensiveproliferation. Because the differences in clonogenicity among theleukemia cells mirrored the differences in clonogenicity among normalhematopoietic cells, the clonogenic leukemic cells were described asleukemic stem cells. It has also been shown for solid cancers that thecells are phenotypically heterogeneous and that only a small proportionof cells are clonogenic in culture and in vivo. Just as in the contextof leukemic stem cells, these observations led to the hypothesis thatonly a few cancer cells are actually tumorigenic and that thesetumorigenic cells act as cancer stem cells.

In support of this hypothesis, recent studies have shown that, similarto leukemia and other hematologic malignancies, tumorigenic andnon-tumorigenic populations of breast cancer cells can be isolated basedon their expression of cell surface markers. In many cases of breastcancer, only a small subpopulation of cells had the ability to form newtumors. This work strongly supports the existence of CSC in breastcancer. Further evidence for the existence of cancer stem cellsoccurring in solid tumors has been found in central nervous system (CNS)malignancies. Using culture techniques similar to those used to culturenormal neuronal stem cells it has been shown that neuronal CNSmalignancies contain a small population of cancer cells that areclonogenic in vitro and initiate tumors in vivo, while the remainingcells in the tumor do not have these properties.

Stem cells are defined as cells that have the ability to perpetuatethemselves through self-renewal and to generate mature cells of aparticular tissue through differentiation. In most tissues, stem cellsare rare. As a result, stem cells must be identified prospectively andpurified carefully in order to study their properties. Perhaps the mostimportant and useful property of stem cells is that of self-renewal.Through this property, striking parallels can be found between stemcells and cancer cells: tumors may often originate from thetransformation of normal stem cells, similar signaling pathways mayregulate self-renewal in stem cells and cancer cells, and cancers maycomprise rare cells with indefinite potential for self-renewal thatdrive tumorigenesis.

The presence of cancer stem cells has profound implications for cancertherapy. At present, all of the phenotypically diverse cancer cells in atumor are treated as though they have unlimited proliferative potentialand can acquire the ability to metastasize. For many years, however, ithas been recognized that small numbers of disseminated cancer cells canbe detected at sites distant from primary tumors in patients that nevermanifest metastatic disease. One possibility is that immune surveillanceis highly effective at killing disseminated cancer cells before they canform a detectable tumor. Another possibility is that most cancer cellslack the ability to form a new tumor such, that only the disseminationof rare cancer stem cells can lead to metastatic disease. If so, thegoal of therapy must be to identify and kill this cancer stem cellpopulation.

The prospective identification and isolation of cancer stem cells willallow more efficient identification of diagnostic markers andtherapeutic targets expressed by the stem cells. Existing therapies havebeen developed largely against the bulk population of tumor cells,because the therapies are identified by their ability to shrink thetumor mass. However, because most cells within a cancer have limitedproliferative potential, an ability to shrink a tumor mainly reflects anability to kill these cells. Therapies that are more specificallydirected against cancer stem cells may result in more durable responsesand cures of metastatic tumors.

Hematopoiesis proceeds through an organized developmental hierarchyinitiated by hematopoietic stem cells (HSC) that give rise toprogressively more committed progenitors and eventually terminallydifferentiated blood cells (Bryder et al., 2006). Although the conceptof the HSC was not new, it was not until 1988 that it was shown thatthis population could be prospectively isolated from mouse bone marrowon the basis of cell-surface markers using fluorescence-activated cellsorting (FACS) (Spangrude et al., 1988). Since that time, the surfaceimmunophenotype of the mouse HSC has become increasingly refined, suchthat functional HSC can be isolated with exquisite sensitivity,resulting in a purity of 1 in 1.3 cells (Kiel et al., 2005). While ourability to prospectively isolate mouse HSC has improved dramaticallyover the past 20 years, our understanding of the earliest events in thehuman hematopoietic system lags far behind.

Cancer stem cells are discussed in, for example, Pardal et al. (2003)Nat Rev Cancer 3, 895-902; Reya et al. (2001) Nature 414, 105-11; Bonnet& Dick (1997) Nat Med 3, 730-7; Al-Hajj et al. (2003) Proc Natl Acad SciUSA 100, 3983-8; Dontu et al. (2004) Breast Cancer Res 6, R605-15; Singhet al. (2004) Nature 432, 396-401.

SUMMARY OF THE INVENTION

Markers of acute myeloid leukemia stem cells (AMLSC) are providedherein. The markers are polynucleotides or polypeptides that aredifferentially expressed on AMLSC as compared to normal counterpartcells. Uses of the markers include use as targets for therapeuticantibodies or ligands; as targets for drug development, and foridentification or selection of AMLSC cell populations.

The AMLSC markers are useful as targets of therapeutic monoclonalantibodies for treatment of patients with de novo, relapsed, orrefractory acute myeloid leukemia. Such monoclonal antibodies are alsouseful in the treatment of pre-leukemic conditions, such asmyelodysplastic syndromes (MDS) and myeloproliferative disorders (MPDs)including: chronic myelogenous leukemia, polycythemia vera, essentialthrombocytosis, agnogenic myelofibrosis and myeloid metaplasia, andothers. Antibodies include free antibodies and antigen binding fragmentsderived therefrom, and conjugates, e.g. pegylated antibodies, drug,radioisotope, or toxin conjugates, and the like.

In some embodiments, combinations of monoclonal antibodies are used inthe treatment of human AML or pre-leukemic conditions. In oneembodiment, a monoclonal antibody directed against CD47, for example anantibody that blocks the interaction of CD47 with SIRPα, is combinedwith monoclonal antibodies directed against one or more additional AMLSCmarkers, e.g. CD96, CD97, CD99, PTHR2, HAVCR2, and the like, whichcompositions can be synergistic in enhancing phagocytosis andelimination of AML LSC as compared to the use of single antibodies.

The AMLSC markers are useful as targets of monoclonal antibodies for usein ex vivo purging of autologous stem cell products (mobilizedperipheral blood or bone marrow) for use in autologous transplantationfor patients with acute myeloid leukemia or the pre-leukemic conditionsoutlined above. Combinations of monoclonal antibodies directed againstAML LSC-specific cell surface molecules, as described above, can besynergistic in eliminating LSC.

The AMLSC markers are useful in clinical diagnostic applicationsincluding, without limitation, primary diagnosis of AML or pre-leukemicconditions from blood and/or bone marrow specimens, evaluation ofleukemic involvement of the cerebrospinal and other body fluids,monitoring of interval disease progression, and monitoring of minimalresidual disease status.

As an alternative to monoclonal antibodies, the ligands of AMLSCmarkers, either as single agents or in combination, may be used totarget them in AML or the pre-leukemic conditions outlined above. Theligands can be free or conjugated, for direct administration to patientsor for ex vivo purging of autologous stem cell products. Some specificmolecules and their ligands include, without limitation, CD155-Fc fusionprotein that binds CD96; TIP39 that binds PTHR2; Galectin-9 that bindsHAVCR2.

The AMLSC cells can be prospectively isolated or identified from primarytumor samples, and possess the unique properties of cancer stem cells infunctional assays for cancer stem cell self-renewal and differentiation.

In some embodiments of the invention, methods are provided fordetection, classification or clinical staging of acute myeloid leukemiasaccording to the stem cells that are present in the leukemia, wheregreater numbers of stem cells are indicative of a more aggressive cancerphenotype. Staging is useful for prognosis and treatment. In someembodiments, a tumor sample is analyzed by histochemistry, includingimmunohistochemistry, in situ hybridization, and the like, for thepresence of CD34⁺CD38⁻ cells that express one or more AMLSC markersprovided herein. The presence of such cells indicates the presence ofAMLSC.

In another embodiment of the invention, methods for the isolation ofAMLSC are provided, comprising contacted a candidate cell populationwith a binding reagent specific for one or more of the AMLSC markersprovided herein, and selecting for cells that have bound to thereagent(s). The cells may further be selected as being CD34⁺CD38⁻. Thecells are useful for experimental evaluation, and as a source of lineageand cell specific products, including mRNA species useful in identifyinggenes specifically expressed in these cells, and as targets for thediscovery of factors or molecules that can affect them. AMLSC may beused, for example, in a method of screening a compound for an effect onthe cells. This involves combining the compound with the cell populationof the invention, and then determining any modulatory effect resultingfrom the compound. This may include examination of the cells forviability, toxicity, metabolic change, or an effect on cell function.The phenotype of AMLSC described herein provides a means of predictingdisease progression, relapse, and development of drug resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Identification of CD90/CD45RA Subpopulations of Lin−CD34+CD38−Human Bone Marrow and Cord Blood. Normal human bone marrow (top) andcord blood (bottom) were analyzed for expression of lineage markers,CD34, CD38, CD90, and CD45RA by flow cytometry. The bone marrow samplewas CD34-enriched prior to analysis. The left panels are gated onlineage negative (Lin−) live cells, while the right panels are gated onLin−CD34+CD38− cells. Data shown is representative of multiple samplesof bone marrow (n=10) and cord blood (n=22).

FIG. 2: In Vitro Evaluation of the CD90/CD45RA Subpopulations ofLin−CD34+CD38− Cord Blood Reveals a Developmental Hierarchy. A.Methylcellulose colony formation. Single cells from each CD90/CD45RAsubpopulation were sorted into individual wells of a 96 well platecontaining complete methylcellulose capable of supporting growth of alltypes of myeloid colonies. After 12-14 days, colonies were scored basedon morphology. The percent of each type of colony out of the total cellsplated is indicated. Data presented is cumulative from 3 experiments of60 cells each, for a total of 180 cells per subpopulation. B.Methylcellulose colony replating. All colonies derived from individualcells were harvested, dissociated, and replated in completemethylcellulose. 12-14 days later, the formation of new colonies wasscored based on morphology. 42 out of 62 (70%) of CD90+CD45RA−, 23 outof 70 (33%) of CD90−CD45RA−, and 0 out of 6 (0%) of CD90−CD45RA+colonies formed new colonies upon replating. The difference in replatingefficiency between CD90+CD45RA− and CD90−CD45RA− was statisticallysignificant (p=0.003). Data presented is the average of 3 independentexperiments with the indicated SEM. C. In vitro proliferation. 20 cellsof each CD90/CD45RA subpopulation were clone sorted into individualwells of a 96 well plate containing serum-free media supplemented withhuman LDL and cytokines. After 2 weeks in culture, cells were harvestedand live cells counted by trypan blue exclusion. The difference betweenthe CD90+CD45RA− and the CD90−CD45RA− subpopulations was statisticallysignificant (p=0.007). Data is representative of 3 independentexperiments. D. In vitro hierarchical relationships among CD90/CD45RAsubpopulations. CD90/CD45RA subpopulations were sorted in bulk intoserum-free media supplemented with human LDL and cytokines. Cells werecultured for four days and then re-analyzed by flow cytometry. All plotsshown are gated on Lin−CD34+CD38− cells; the left panels show the cellinput; the right panels show the cells after four days in culture. Datashown is representative of 3 independent experiments.

FIG. 3: Long-Term In Vivo Multipotent Human Hematopoiesis withTransplantation of as few as 10 Lin−CD34+CD38−CD90+CD45RA− Cord BloodCells. A. In vivo engraftment of 100 or 10 CD90+CD45RA− cells. 100 or 10FACS-purified CD90+CD45RA− cells were transplanted into NOG newborn miceas described. 12 weeks later peripheral blood and/or bone marrow washarvested and analyzed by flow cytometry for the presence of human CD45+hematopoietic cells, myeloid cells (hCD45+CD13+), and B lymphoid cells(hCD45+CD19+) cells. The right plots are gated on human CD45+ cells. B.Wright-Giemsa stained cytosine preparations from CD90+CD45RA− engraftedmice. Human CD45+ cells were purified by FACS from peripheral blood(panels 1-3) or bone marrow (panel 4) of mice engrafted withCD90+CD45RA− cells. In the peripheral blood, (1) lymphocytes, (2)neutrophils, and (3) monocytes were detected; in the bone marrow (4)lymphocytes and maturing myeloid cells were detected. (100×)

FIG. 4: Human Lymphoid and Myeloid Cells Reconstitute the PeripheralBlood of CD90/CD45RA Transplanted Mice. A. Peripheral blood engraftmentand lineage analysis of CD90/CD45RA transplanted mice. 500 FACS-purifiedcells of each CD90/CD45RA subpopulation: CD90+CD45RA− (top panels),CD90−CD45RA− (middle panels), and CD90−CD45RA+(bottom panels), weretransplanted into NOG newborn mice as described. 12 weeks laterperipheral blood was harvested and analyzed by flow cytometry for thepresence of human CD45+ hematopoietic cells, myeloid cells(hCD45+CD13+), and B lymphoid cells (hCD45+CD19+) cells. The right plotsare gated on human CD45+ cells. B. Summary of long-term (>12 weeks)peripheral blood engraftment of CD90/CD45RA subpopulations. C.Peripheral blood engraftment per 100 transplanted cells. The percenthuman chimerism (left) and percent human myeloid cells (right) per 100transplanted cells is indicated for each engrafted mouse. Each circle ortriangle represents an individual mouse and the bar indicates theaverage. On average, CD90+CD45RA− mice developed 7 fold more humanchimerism than CD90−CD45RA− mice, and this difference was statisticallysignificant (p=0.02). The 7 fold difference in percent human myeloidcells approached, but did not achieve statistical significance (p=0.08).

FIG. 5: Human Lymphoid and Myeloid Cells Reconstitute the Bone Marrow ofCD90+CD45RA− Transplanted Mice More Efficiently than CD90−CD45RA−Transplanted Mice. A. Bone marrow engraftment and lineage analysis ofCD90/CD45RA transplanted mice. 500 FACS-purified CD90+CD45RA− cells (toppanels) and CD90−CD45RA− cells (bottom panels) were transplanted intoNOG newborn mice as described. 12 weeks later bone marrow was analyzedby flow cytometry for the presence of human CD45+ hematopoietic cells,myeloid cells (hCD45+CD33+), and B lymphoid cells (hCD45+CD19+) cells.The right plots are gated on human CD45+ cells. B. Summary of long-term(>12 weeks) bone marrow engraftment of CD90/CD45RA subpopulations. C.Bone marrow engraftment per 100 transplanted cells. The percent humanchimerism (left) and percent human myeloid cells (right) per 100transplanted cells is indicated for each engrafted mouse. Each circle ortriangle represents an individual mouse and the bar indicates theaverage. On average, CD90+CD45RA− mice developed 9 fold more humanchimerism than CD90−CD45RA− mice, and this difference was statisticallysignificant (p=0.001). The 9 fold difference in percent human myeloidcells approached, but did not achieve statistical significance (p=0.07).D. Summary of long-term (>11 weeks) bone marrow engraftment of limitingnumbers (<100 cells) of CD90/CD45RA subpopulations. 50 or 70 doubleFACS-purified CD90+CD45RA− or CD90−CD45RA− cells were transplanted intoNOG newborn mice as described. At least 11 weeks later bone marrow wasanalyzed by flow cytometry for the presence of human CD45+ hematopoieticcells, myeloid cells (hCD45+CD33+), B cells (hCD45+CD19+) cells, and Tcells (hCD45+CD3+). In 1 out of 5 mice transplanted with CD90−CD45RA−cells, a small population of T cells (0.2%) was detected in the bonemarrow, in the absence of myeloid and B cells. The difference insuccessful engraftment was statistically significant (p=0.008).

FIG. 6: In Vivo Analysis of Human CD34+ Cells Identifies a HierarchyAmong the CD90/CD45RA Subpopulations. A. Summary of long-term (>12weeks) human CD34+ bone marrow engraftment of CD90/CD45RAsubpopulations. B. Bone marrow human CD34+ engraftment per 100transplanted cells. The percentage of human CD34+ cells in total bonemarrow per 100 transplanted cells is indicated for each engrafted mouse.Each circle represents an individual mouse and the bar indicates theaverage. On average, CD90+CD45RA− mice contained 8 fold more human CD34+cells than CD90−CD45RA− mice, and this difference was statisticallysignificant (p=0.01). C. Analysis of CD90/CD45RA expression onLin−CD34+CD38− bone marrow cells in CD90/CD45RA transplanted mice. 500FACS-purified CD90+CD45RA− cells (top panels) and CD90−CD45RA− cells(bottom panels) were transplanted into NOG newborn mice as described. 12weeks later bone marrow was analyzed by flow cytometry for theexpression of lineage markers, CD34, CD38, CD90, and CD45RA. The leftplots are gated on Lin−CD34+ cells, and the right plots are gated onLin−CD34+CD38− cells. D. CD90/CD45RA subpopulations within engraftedbone marrow. The percentage of CD90+CD45RA− (left), CD90−CD45RA−(middle), and CD90−CD45RA+(right) cells out of Lin−CD34+CD38− bonemarrow cells from mice engrafted with CD90+CD45RA− and CD90−CD45RA−cells is indicated. Each circle, triangle, or square represents anindividual mouse. Only mice with greater than 10 Lin−CD34+CD38− cellswere included (n=7 transplanted with CD90+CD45RA− cells and n=4transplanted with CD90−CD45RA− cells).

FIG. 7: Enrichment of Secondary Transplant Ability in the CD90+CD45RASubpopulation. A. Summary of long-term (>10 weeks) bone marrowengraftment in secondary transplants from mice engrafted with eitherCD90+ or CD90− subpopulations. The difference in successful secondaryengraftment, 12 of 12 (100%) for CD90+ versus 3 of 8 (37.5%) for CD90−,was statistically significant with p=0.004. B. Bone marrow engraftmentin secondary recipients from experiment 1. Primary mice weretransplanted with 2000 cells of the indicated population. The percenthuman chimerism (left) and percent myeloid cells of total human CD45+cells (right) is indicated for each secondary mouse. Each circle ortriangle represents an individual mouse. C. Bone marrow engraftment insecondary recipients from experiment 2. Primary mice were transplantedwith 500 cells of the indicated population. The percent human chimerism(left) and percent myeloid cells of total human CD45+ cells (right) isindicated for each secondary mouse. Each circle or triangle representsan individual mouse.

FIG. 8: Differential Gene Expression Between AML LSC and Normal BoneMarrow HSC and MPP (A) Heat maps demonstrating genes found to bedifferentially expressed at least 2 fold between bone marrow HSC (n=4)and AML LSC (n=9) or bone marrow MPP (n=4) and AML LSC (n=9). Expressionrelative to the median is indicated for genes with p<0.05 and a FDR of5%. (B) Selected list of transmembrane proteins found to be at least2-fold more highly expressed in AML LSC than HSC of MPP. NS: notsignificant.

FIG. 9. CD47 is more highly expressed on AML LSC. Mobilized peripheralblood (MPB) HSC and AML LSC were examined for CD47 expression by flowcytometry. (A) Representative flow cytometry plots indicating expressionof CD47 relative to an isotype control. (B) Summary of CD47 expressionon all samples assayed, with the indicated means.

FIG. 10: Anti-CD47 Antibody Stimulates In Vitro Macrophage Phagocytosisof Primary Human AML LSC. AML LSC were purified by FACS from two primaryhuman AML samples, labeled with the fluorescent dye CFSE, and incubatedwith mouse bone marrow-derived macrophages either in the presence of anisotype control (A) or anti-CD47 antibody (B). These cells were assessedby immunofluorescence microscopy for the presence of fluorescentlylabeled LSC within the macrophages. (C) The phagocytic index wasdetermined for each condition by calculating the number of ingestedcells per 100 macrophages.

FIG. 11. Anti-CD47 antibody stimulates in vitro macrophage phagocytosisof primary human AML LSC. AML LSC were purified by FACS from two primaryhuman AML samples and labeled with the fluorescent dye CFSE. These cellswere incubated with mouse bone marrow-derived macrophages, either in thepresence of an isotype matched control (left) or anti-CD47 antibody(right). The macrophages were harvested, stained with a fluorescentlylabeled anti-mouse macrophage antibody, and analyzed by flow cytometry.mMac+CFSE+ double-positive events identify macrophages that havephagocytosed CFSE-labeled LSC. (A,B) two independent primary AML LSCsamples.

FIG. 12. Anti-CD47 antibody inhibits in vivo engraftment of primaryhuman AML. Two primary human AML samples were untreated (control, n=3)or coated with anti-human CD47 antibody (anti-CD47, n=6) prior totransplantation into newborn NOG mice. 13 weeks later, mice weresacrificed and the bone marrow was analyzed for the presence of humanCD45+CD33+ myeloid leukemia cells by flow cytometry.

FIG. 13. CD99 is highly expressed on AML LSC. Cord blood (CB) HSC andAML LSC were examined for CD99 expression by flow cytometry. (A)representative flow cytometry plots indicating expression of CD99relative to an isotype matched control. (B) summary of CD99 expressionon all samples assayed.

FIG. 14. CD97 is preferentially expressed on LSC. Cord blood HSC(Lin−CD34+CD38− CD90+, n=3) and AML LSC (Lin−CD34+CD38−CD90+, n=7) wereexamined for CD97 expression by flow cytometry. (A) representative flowcytometry plots indicating expression of CD97 relative to an isotypematched control. (B) Summary of CD97 expression on all samples assayed.

FIG. 15. CD47 is upregulated in murine acute myeloid leukemia. Typicalstem and progenitor plots are shown for leukemic hMRP8bcrabl×hMRP8bcl2cells compared to control non-leukemic animals. Lin− c-Kit+ Sca-1+ gatedcells from control bone marrow (a) and leukemic spleen (b) and Lin−c-Kit+ Sca-1− gated cells from control bone marrow (c) and leukemicspleen (d) demonstrate perturbances in normal haematopoiesis in leukemicmice. Frequency is shown as a percentage of entire marrow or spleenmononuclear fraction. (e) Quantitative RT-PCR shows that CD47 isupregulated in leukemic BM cells. Data are shown from 3 sets of micetransplanted with either leukemic or control hRMP8bcrabl×hMRP8bcl2 BMcells and then sacrificed 2-6 weeks later. Results were normalized tobeta-actin and 18S rRNA expression. Fold change relative to controltransplanted whole Bcl-2+ BM cells was determined. Error bars represent1 s.d. (f) Histograms show expression of CD47 on gated populations forleukemic (gray) and control (black) mice.

FIG. 16. GMP expansion and CD47 upregulation in human myeloid leukemia.a) Representative FACS plots of myeloid progenitors (CD34+CD38+Lin−)including common myeloid progenitors (CMP), megakaryocyte-erythroidprogenitors (MEP) and granulocyte-macrophage progenitors (GMP) in normalbone marrow (BM) versus aCML, BC CML and AML. b) Comparative FACShistograms of CD47 expression by normal (red; n=6) and acute myelogenousleukemic (AML, blue; n=6) haematopoietic stem cells (HSC;CD34+CD38−CD90+Lin−) and progenitors (CD34+CD38+Lin−). c) ComparativeFACS histograms of CD47 expression by normal (red) and chronicmyelogenous leukemia haematopoietic stem cells (HSC; CD34+CD38−CD90+Lin)and committed progenitors (CD34+CD38+Lin−). Upper panel: Normal (n=7)versus chronic phase CML (n=4) HSC, progenitors and lineage positivecells. Middle panel: Normal (n=7) versus accelerated phase CML (n=7)HSC, progenitors and lineage positive cells. Lower panel: Normal (n=7)versus blast crisis CML (n=4) HSC, progenitors and lineage positivecells.

FIG. 17. Over-expression of murine CD47 increases tumorigenicity ofMOLM-13 cells. a) MOLM-13 cells were transduced with either controlvirus or virus expressing murine CD47 cDNA form 2. The resulting celllines, termed Tet or Tet-CD47, were transplanted competitively intoRAG/common gamma chain deficient mice with untransduced MOLM-13 cells(5×10⁵ Tet (n=6) or Tet-47 (n=8) cells with 5×10⁵ MOLM-13). Mice wereanalyzed for GFP and human CD45 chimerism when moribund. b) MOLM-13chimerism in haematopoietic tissues was determined by human CD45chimerism and measurement of tumour lesion size. c) Survival of micecompetitively transplanted with MOLM-13 plus Tet or Tet-CD47 MOLM-13cells was plotted. Control mice died of large tumour burden at the siteof injection but had no engraftment in haematopoietic tissues. d)Hematoxylin and eosin sections of Tet-CD47 MOLM-13 transplanted liver(200×). Periportal (arrow) and sinusoidal (arrowhead) tumor infiltrationis evident. e) 1×10⁶ Tet (n=5) or Tet-CD47 MOLM-13 (n=4) cells wereinjected into the right femur of RAG2−/−, Gc−/− mice and the tissueswere analyzed 50-75 days later and chimerism of MOLM-13 cells in bonemarrow was determined. f) Survival curve of mice transplantedintrafemorally with Tet or Tet-CD47 MOLM-13 cells. g) Examples of livertumour formation and hepatomegaly in Tet-CD47 MOLM-13 transplanted miceversus control transplanted mice. GFP fluorescence demonstrates tumournodule formation as well diffuse infiltration.

FIG. 18. CD47 over-expression prevents phagocytosis of live unopsonizedMOLM-13 cells. a) Tet or Tet-CD47 MOLM-13 cells were incubated with bonemarrow derived macrophages (BMDM) for 2, 4, or 6 hours and phagocyticindex was determined. Error bars represent 1 s.d (n=6 for each timepoint). b) FACS analysis of BMDMs incubated with either Tet or Tet-CD47cells. c) Photomicrographs of BMDMs incubated with Tet or Tet-CD47MOLM-13 cells at 2 and 24 hours (400×). d) Tet or Tet-CD47 MOLM-13 cellswere transplanted into RAG2−/−, Gc−/− mice and marrow, spleen, and livermacrophages were analyzed 2 hours later. GFP+ fraction of macrophagesare gated. Results are representative of 3 experiments.

FIG. 19. Higher expression of CD47 on MOLM-13 cells correlates withtumorigenic potential and evasion of phagocytosis. a) Tet-CD47 MOLM-13cells were divided into high and low expressing clones as described.Histograms show CD47 expression in MOLM-13 high (black), MOLM-13 low(gray), and mouse bone marrow (shaded) cells. Value obtained forMFI/FSC² (×10⁹) are shown. b) Mice transplanted with CD47hi MOLM-13cells were given doxycycline for 2 weeks. The histograms show level ofCD47 expression in untreated (shaded) and treated (shaded) mice, withthe values of MFI/FSC² (×10⁹) indicated. c) Survival of RAG2−/−, Gc−/−mice transplanted with 1×10⁶ CD47^(hi), CD47^(lo) MOLM-13 cells, orCD47^(hi) MOLM-13 cells with doxycycline administration after 2 weekspost-transplant. d) Liver and spleen size of mice at necropsy or 75 daysafter transplant with 1×10⁶ CD47^(hi), CD47^(lo) MOLM-13 cells, orCD47^(hi) MOLM-13 cells with doxycycline administration after 2 weekspost-transplant. e) Bone marrow and spleen chimerism of human cells inmice at necropsy or 75 days after transplant with 1×10⁶ CD47^(hi),CD47^(lo) MOLM-13 cells, or CD47^(loi) MOLM-13 cells with doxycyclineadministration after 2 weeks post-transplant. f) Murine CD47 expressionon CD47l^(o) MOLM-13 cells engrafting in bone marrow (open) comparedwith original cell line (shaded). The values of MFI/FSC² (×10⁹) areindicated. g) 2.5×10⁵ CD47^(hi) or CD47^(lo) MOLM-13 cells wereincubated with 5×10⁴ BMDMs for 2 hours. Phagocytic index is shown. h)2.5×10⁵ CD47^(hi) RFP and CD47^(lo) MOLM-13 GFP cells were incubatedwith 5×10⁴ BMDMs for 2 hours. Phagocytic index is shown for threeseparate samples for CD47^(hi) RFP (red) and CD47^(lo) MOLM-13 GFP(green) cells. i) 2.5×10⁵ CD47^(hi) RFP and CD47^(lo) MOLM-13 GFP cellswere incubated with 5×10⁴ BMDMs for 24 hours. Photomicrographs showbrightfield (top left), RFP (top right), GFP (bottom left), and merged(bottom right) images.

FIG. 20. a) FACS analysis of CD47 expression of non-leukemic Fas lpr/lprhMRP8bcl-2 (blue) and leukemic Fas lpr/lpr hMRP8bcl-2 (green) bonemarrow haematopoietic stem cells (c-kit+Sca+Lin−), myeloid progenitors(c-kit+Sca−Lin−) or blasts (c-kit lo Sca−Lin−). b) Mouse bone marrow wastransduced with retrovirus containing p210 bcr/abl as previouslydescribed²⁴. Mice were sacrificed when moribund and the spleens wereanalyzed. Expression of CD47 in c-Kit+Mac−1+ cells in the spleens of twoleukemic mice (unshaded histograms) and bone marrow from a wild-typemouse (shaded histogram) are shown. c) Histograms show expression ofCD47 on gated populations for leukemic hMRP8bcrabl×hMRP8bcl2 mice (red),hMRP8bcl2 non-leukemic (blue) and wild-type (green) mice. CD47 wasstained using FITC conjugated anti-mouse CD47 (Pharmingen).

FIG. 21. a) Mean % of CD34+CD38+Lin− progenitors in normal versuspolycythemia vera (PV; n=16), post-polycythemic myeloidmetaplasia/myelofibrosis (PPMM/MF; n=5), essential thrombocythemia (ET;n=7), chronic myelomonocytic leukemia (CMML; n=11), atypical chronicmyelogenous leukemia (aCML; n=1), blast crisis CML (BC CML; n=6) andacute myelogenous leukemia (AML; n=13) peripheral blood or bone marrow.b) Staining for CD47 using newer staining protocol on stem andprogenitor cells from normal human cord blood, chronic phase, and blastcrisis CML. Normal (green), CML-CP (red), and CML-BC (blue) are shown.

FIG. 22. CD123/CD45a FACS plot of normal marrow (left), acceleratedphase CML (middle) and CML BC progenitors (right).

FIG. 23. Representative FACS plots from mice transplanted intrafemorallywith Tet or Tet-CD47 MOLM-13 cells. All 6 long bones as well as spleendemonstrate profound MOLM-13 engraftment in Tet-CD47 transplanted mice.R-right, L-left.

FIG. 24. a) Expression of human CD47 (black histograms) on humanleukemia cell lines and cord blood HSCs is shown. Isotype controlstaining is shown in gray. b) CD47 MFI over background was normalized tocell size by dividing by FSC². The value obtained for each cell type isshown above the bar. c) HL-60 cells engraft mouse bone marrow. 5×10⁵cells were injected intravenously into RAG2−/−, Gc−/− animals and micewere analyzed 4 weeks later. d) Cells were stained with CFSE andco-cultured with BMDM. Phagocytic events were counted after 2 h. Forirradiation, Jurkat cells were given a dose of 2 Gray and incubated for16 h prior to the phagocytosis assay.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention identifies polynucleotides, as well aspolypeptides encoded thereby, that are differentially expressed in acutemyeloid leukemia stem cells (AMLSC). Methods are provided in which thesepolynucleotides and polypeptides, which may be collectively referred toas AMLSC markers, are used for detecting, assessing, and reducing thegrowth of cancer cells. Methods may use one or a combination of markers,where a combination may include 2, 3 or more markers, and in someembodiments will include CD47 in combination with 1, 2 or more markers.The invention finds use in the prevention, treatment, detection orresearch of leukemic and pre-leukemic conditions.

The markers of the invention in some embodiments are expressed on theAMLSC cell surface. In some embodiments, the markers are expressed as alevel at least 2× the expression level of a counterpart non-transformedcell, e.g. a human hematopoietic stem cell, and/or a human hematopoieticmultipotent progenitor cell, where expression may be determined as thelevel of transcription, mRNA accumulation, and/or protein accumulation.In other embodiments the markers are expressed as a level at least 3×,at least 4×, at least 5×, at least 10×, at least 20× or greater, thanthe expression level of a counterpart non-transformed cell.

The present invention provides methods of using the markers describedherein in diagnosis of cancer, classification and treatment of leukemicand pre-leukemic conditions according to expression profiles. Themethods are useful for detecting AMLSC, facilitating diagnosis of AMLand the severity of the cancer (e.g., tumor grade, tumor burden, and thelike) in a subject, facilitating a determination of the prognosis of asubject, and assessing the responsiveness of the subject to therapy. Thedetection methods of the invention can be conducted in vitro or in vivo,on isolated cells, or in whole tissues or a bodily fluid, e.g., blood,lymph node biopsy samples, and the like.

As used herein, the terms “a gene that is differentially expressed in acancer stem cell,” and “a polynucleotide that is differentiallyexpressed in a cancer stem cell”, are used interchangeably herein, andgenerally refer to a polynucleotide that represents or corresponds to agene that is differentially expressed in a cancer stem cell whencompared with a cell of the same cell type that is not cancerous, e.g.,mRNA is found at levels at least about 25%, at least about 50% to about75%, at least about 90%, at least about 1.5-fold, at least about 2-fold,at least about 3-fold, at least about 5-fold, at least about 10-fold, orat least about 50-fold or more, different (e.g., higher or lower). Thecomparison can be made between AMLSC and the normal counterpart cells ahuman hematopoietic stem cell (HSC), which include without limitationcells having the phenotype Lin⁻CD34⁺CD38⁻CD90⁺; or the phenotypeLin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻ and a human hematopoietic multipotentprogenitor cell (MPP), which include without limitation cells having thephenotype Lin⁻CD34⁺CD38⁻CD90⁻; or the phenotypeLin⁻CD34⁺CD38⁻CD90⁻CD45RA⁻. The term “a polypeptide marker for a cancerstem cell” refers to a polypeptide encoded by a polynucleotide that isdifferentially expressed in a cancer stem cell.

In some embodiments of the invention, the markers are demonstrated byflow cytometry to be present on a majority of AMLSC, when compared tohuman HSC or MPP, as defined above. Such markers include, withoutlimitation, CD47, CD96, CD97 and CD99.

In other embodiments of the invention, the markers are absent on humanHSC or human MPP, but are highly expressed on AMLSC. Such markersinclude, without limitation, those set forth in Table 2.

In other embodiments, the markers are differentially expressed on AMLSC,as compared to human HSC or MPP. Such markers include, withoutlimitation, those set forth in Table 3.

A polynucleotide or sequence that corresponds to, or represents a genemeans that at least a portion of a sequence of the polynucleotide ispresent in the gene or in the nucleic acid gene product (e.g., mRNA orcDNA). A subject nucleic acid may also be “identified” by apolynucleotide if the polynucleotide corresponds to or represents thegene. Genes identified by a polynucleotide may have all or a portion ofthe identifying sequence wholly present within an exon of a genomicsequence of the gene, or different portions of the sequence of thepolynucleotide may be present in different exons (e.g., such that thecontiguous polynucleotide sequence is present in an mRNA, either pre- orpost-splicing, that is an expression product of the gene). An“identifying sequence” is a minimal fragment of a sequence of contiguousnucleotides that uniquely identifies or defines a polynucleotidesequence or its complement.

The polynucleotide may represent or correspond to a gene that ismodified in a cancer stem cell (CSC) relative to a normal cell. The genein the CSC may contain a deletion, insertion, substitution, ortranslocation relative to the polynucleotide and may have alteredregulatory sequences, or may encode a splice variant gene product, forexample. The gene in the CSC may be modified by insertion of anendogenous retrovirus, a transposable element, or other naturallyoccurring or non-naturally occurring nucleic acid.

Sequences of interest include those set forth in Table 1, which aredifferentially expressed in AMLSC relative to normal counterpart cells.

Methods are also provided for optimizing therapy, by firstclassification, and based on that information, selecting the appropriatetherapy, dose, treatment modality, etc. which optimizes the differentialbetween delivery of an anti-proliferative treatment to the undesirabletarget cells, while minimizing undesirable toxicity. The treatment isoptimized by selection for a treatment that minimizes undesirabletoxicity, while providing for effective anti-proliferative activity.

The invention finds use in the prevention, treatment, detection orresearch of acute myeloid leukemias. Acute leukemias are rapidlyprogressing leukemia characterized by replacement of normal bone marrowby blast cells of a clone arising from malignant transformation of ahematopoietic stem cell. The acute leukemias include acute lymphoblasticleukemia (ALL) and acute myelogenous leukemia (AML). ALL often involvesthe CNS, whereas acute monoblastic leukemia involves the gums, and AMLinvolves localized collections in any site (granulocytic sarcomas orchloromas). AML is the most common acute leukemia affecting adults, andits incidence increases with age. While AML is a relatively rare diseaseoverall, accounting for approximately 1.2% of cancer deaths in theUnited States, its incidence is expected to increase as the populationages.

The presenting symptoms are usually nonspecific (e.g., fatigue, fever,malaise, weight loss) and reflect the failure of normal hematopoiesis.Anemia and thrombocytopenia are very common (75 to 90%). The WBC countmay be decreased, normal, or increased. Blast cells are usually found inthe blood smear unless the WBC count is markedly decreased. The blastsof ALL can be distinguished from those of AML by histochemical studies,cytogenetics, immunophenotyping, and molecular biology studies. Inaddition to smears with the usual stains, terminal transferase,myeloperoxidase, Sudan black B, and specific and nonspecific esterase.

“Diagnosis” as used herein generally includes determination of asubject's susceptibility to a disease or disorder, determination as towhether a subject is presently affected by a disease or disorder,prognosis of a subject affected by a disease or disorder (e.g.,identification of cancerous states, stages of cancer, or responsivenessof cancer to therapy), and use of therametrics (e.g., monitoring asubject's condition to provide information as to the effect or efficacyof therapy).

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

A “host cell”, as used herein, refers to a microorganism or a eukaryoticcell or cell line cultured as a unicellular entity which can be, or hasbeen, used as a recipient for a recombinant vector or other transferpolynucleotides, and include the progeny of the original cell which hasbeen transfected. It is understood that the progeny of a single cell maynot necessarily be completely identical in morphology or in genomic ortotal DNA complement as the original parent, due to natural, accidental,or deliberate mutation.

The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, are usedinterchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation. Ingeneral, cells of interest for detection or treatment in the presentapplication include precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and non-metastatic cells. Detection ofcancerous cells is of particular interest. The term “normal” as used inthe context of “normal cell,” is meant to refer to a cell of anuntransformed phenotype or exhibiting a morphology of a non-transformedcell of the tissue type being examined. “Cancerous phenotype” generallyrefers to any of a variety of biological phenomena that arecharacteristic of a cancerous cell, which phenomena can vary with thetype of cancer. The cancerous phenotype is generally identified byabnormalities in, for example, cell growth or proliferation (e.g.,uncontrolled growth or proliferation), regulation of the cell cycle,cell mobility, cell-cell interaction, or metastasis, etc.

“Therapeutic target” refers to a gene or gene product that, uponmodulation of its activity (e.g., by modulation of expression,biological activity, and the like), can provide for modulation of thecancerous phenotype. As used throughout, “modulation” is meant to referto an increase or a decrease in the indicated phenomenon (e.g.,modulation of a biological activity refers to an increase in abiological activity or a decrease in a biological activity).

Acute Myeloid Leukemia

Acute Myelocytic Leukemia (AML, Acute Myelogenous Leukemia; AcuteMyeloid Leukemia). In AML, malignant transformation and uncontrolledproliferation of an abnormally differentiated, long-lived myeloidprogenitor cell results in high circulating numbers of immature bloodforms and replacement of normal marrow by malignant cells. Symptomsinclude fatigue, pallor, easy bruising and bleeding, fever, andinfection; symptoms of leukemic infiltration are present in only about5% of patients (often as skin manifestations). Examination of peripheralblood smear and bone marrow is diagnostic. Treatment includes inductionchemotherapy to achieve remission and post-remission chemotherapy (withor without stem cell transplantation) to avoid relapse.

AML has a number of subtypes that are distinguished from each other bymorphology, immunophenotype, and cytochemistry. Five classes aredescribed, based on predominant cell type, including myeloid,myeloid-monocytic, monocytic, erythroid, and megakaryocytic. Acutepromyelocytic leukemia is a particularly important subtype, representing10 to 15% of all cases of AML, striking a younger age group (median age31 yr) and particular ethnicity (Hispanics), in which the patientcommonly presents with a coagulation disorder.

Remission induction rates range from 50 to 85%. Long-term disease-freesurvival reportedly occurs in 20 to 40% of patients and increases to 40to 50% in younger patients treated with stem cell transplantation.

Prognostic factors help determine treatment protocol and intensity;patients with strongly negative prognostic features are usually givenmore intense forms of therapy, because the potential benefits arethought to justify the increased treatment toxicity. The most importantprognostic factor is the leukemia cell karyotype; favorable karyotypesinclude t(15;17), t(8;21), and inv16 (p13;q22). Negative factors includeincreasing age, a preceding myelodysplastic phase, secondary leukemia,high WBC count, and absence of Auer rods. The FAB or WHO classificationalone does not predict response.

Initial therapy attempts to induce remission and differs most from ALLin that AML responds to fewer drugs. The basic induction regimenincludes cytarabine by continuous IV infusion or high doses for 5 to 7days; daunorubicin or idarubicin is given IV for 3 days during thistime. Some regimens include 6-thioguanine, etoposide, vincristine, andprednisone, but their contribution is unclear. Treatment usually resultsin significant myelosuppression, with infection or bleeding; there issignificant latency before marrow recovery. During this time, meticulouspreventive and supportive care is vital.

Polypeptide and Polynucleotide Sequences and Antibodies

The invention provides polynucleotides and polypeptides that representgenes that are differentially expressed in human AMLSC. Thesepolynucleotides, polypeptides and fragments thereof have uses thatinclude, but are not limited to, diagnostic probes and primers asstarting materials for probes and primers, as immunogens for antibodiesuseful in cancer diagnosis and therapy, and the like as discussedherein.

Nucleic acid compositions include fragments and primers, and are atleast about 15 bp in length, at least about 30 bp in length, at leastabout 50 bp in length, at least about 100 bp, at least about 200 bp inlength, at least about 300 bp in length, at least about 500 bp inlength, at least about 800 bp in length, at least about 1 kb in length,at least about 2.0 kb in length, at least about 3.0 kb in length, atleast about 5 kb in length, at least about 10 kb in length, at leastabout 50 kb in length and are usually less than about 200 kb in length.In some embodiments, a fragment of a polynucleotide is the codingsequence of a polynucleotide. Also included are variants or degeneratevariants of a sequence provided herein. In general, variants of apolynucleotide provided herein have a fragment of sequence identity thatis greater than at least about 65%, greater than at least about 70%,greater than at least about 75%, greater than at least about 80%,greater than at least about 85%, or greater than at least about 90%,95%, 96%, 97%, 98%, 99% or more (i.e. 100%) as compared to anidentically sized fragment of a provided sequence. as determined by theSmith-Waterman homology search algorithm as implemented in MPSRCHprogram (Oxford Molecular). Nucleic acids having sequence similarity canbe detected by hybridization under low stringency conditions, forexample, at 50° C. and 10×SSC (0.9 M saline/0.09 M sodium citrate) andremain bound when subjected to washing at 55° C. in 1×SSC. Sequenceidentity can be determined by hybridization under high stringencyconditions, for example, at 50° C. or higher and 0.1×SSC (9 mMsaline/0.9 mM sodium citrate). Hybridization methods and conditions arewell known in the art, see, e.g., U.S. Pat. No. 5,707,829. Nucleic acidsthat are substantially identical to the provided polynucleotidesequences, e.g. allelic variants, genetically altered versions of thegene, etc., bind to the provided polynucleotide sequences understringent hybridization conditions.

Probes specific to the polynucleotides described herein can be generatedusing the polynucleotide sequences disclosed herein. The probes areusually a fragment of a polynucleotide sequences provided herein. Theprobes can be synthesized chemically or can be generated from longerpolynucleotides using restriction enzymes. The probes can be labeled,for example, with a radioactive, biotinylated, or fluorescent tag.Preferably, probes are designed based upon an identifying sequence ofany one of the polynucleotide sequences provided herein.

The nucleic acid compositions described herein can be used to, forexample, produce polypeptides, as probes for the detection of mRNA inbiological samples (e.g., extracts of human cells) or cDNA produced fromsuch samples, to generate additional copies of the polynucleotides, togenerate ribozymes or antisense oligonucleotides, and as single strandedDNA probes or as triple-strand forming oligonucleotides.

The probes described herein can be used to, for example, determine thepresence or absence of any one of the polynucleotide provided herein orvariants thereof in a sample. These and other uses are described in moredetail below. In one embodiment, real time PCR analysis is used toanalyze gene expression.

The polypeptides contemplated by the invention include those encoded bythe disclosed polynucleotides and the genes to which thesepolynucleotides correspond, as well as nucleic acids that, by virtue ofthe degeneracy of the genetic code, are not identical in sequence to thedisclosed polynucleotides. Further polypeptides contemplated by theinvention include polypeptides that are encoded by polynucleotides thathybridize to polynucleotide of the sequence listing. Thus, the inventionincludes within its scope a polypeptide encoded by a polynucleotidehaving the sequence of any one of the polynucleotide sequences providedherein, or a variant thereof.

In general, the term “polypeptide” as used herein refers to both thefull length polypeptide encoded by the recited polynucleotide, thepolypeptide encoded by the gene represented by the recitedpolynucleotide, as well as portions or fragments thereof. “Polypeptides”also includes variants of the naturally occurring proteins, where suchvariants are homologous or substantially similar to the naturallyoccurring protein, and can be of an origin of the same or differentspecies as the naturally occurring protein. In general, variantpolypeptides have a sequence that has at least about 80%, usually atleast about 90%, and more usually at least about 98% sequence identitywith a differentially expressed polypeptide described herein. Thevariant polypeptides can be naturally or non-naturally glycosylated,i.e., the polypeptide has a glycosylation pattern that differs from theglycosylation pattern found in the corresponding naturally occurringprotein.

Fragments of the polypeptides disclosed herein, particularlybiologically active fragments and/or fragments corresponding tofunctional domains, are of interest. Fragments of interest willtypically be at least about 10 aa to at least about 15 aa in length,usually at least about 50 aa in length, and can be as long as 300 aa inlength or longer, but will usually not exceed about 1000 aa in length,where the fragment will have a stretch of amino acids that is identicalto a polypeptide encoded by a polynucleotide having a sequence of anyone of the polynucleotide sequences provided herein, or a homologthereof. A fragment “at least 20 aa in length,” for example, is intendedto include 20 or more contiguous amino acids from, for example, thepolypeptide encoded by a cDNA, in a cDNA clone contained in a depositedlibrary or the complementary stand thereof. In this context “about”includes the particularly recited value or a value larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids. The protein variants describedherein are encoded by polynucleotides that are within the scope of theinvention. The genetic code can be used to select the appropriate codonsto construct the corresponding variants. The polynucleotides may be usedto produce polypeptides, and these polypeptides may be used to produceantibodies by known methods described above and below.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides can also be recovered from: products purified from naturalsources, including bodily fluids, tissues and cells, whether directlyisolated or cultured; products of chemical synthetic procedures; andproducts produced by recombinant techniques from a prokaryotic oreukaryotic host, including, for example, bacterial, yeast higher plant,insect, and mammalian cells.

Gene products, including polypeptides, mRNA (particularly mRNAs havingdistinct secondary and/or tertiary structures), cDNA, or complete gene,can be prepared and used for raising antibodies for experimental,diagnostic, and therapeutic purposes. Antibodies may be used to identifyAMLSC cells or subtypes. The polynucleotide or related cDNA is expressedas described herein, and antibodies are prepared. These antibodies arespecific to an epitope on the polypeptide encoded by the polynucleotide,and can precipitate or bind to the corresponding native protein in acell or tissue preparation or in a cell-free extract of an in vitroexpression system.

The antibodies may be utilized for immunophenotyping of cells andbiological samples. The translation product of a differentiallyexpressed gene may be useful as a marker. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al. Cell, 96:737-49 (1999)).These techniques allow for the screening of particular populations ofcells; in immunohistochemistry of biopsy samples; in detecting thepresence of markers shed by cancer cells into the blood and otherbiologic fluids, and the like.

In many embodiments, the levels of a subject gene or gene product aremeasured. By measured is meant qualitatively or quantitativelyestimating the level of the gene product in a first biological sampleeither directly (e.g. by determining or estimating absolute levels ofgene product) or relatively by comparing the levels to a second controlbiological sample. In many embodiments the second control biologicalsample is obtained from an individual not having cancer. As will beappreciated in the art, once a standard control level of gene expressionis known, it can be used repeatedly as a standard for comparison. Othercontrol samples include samples of cancerous tissue.

The methods can be used to detect and/or measure mRNA levels of a genethat is differentially expressed in a cancer cell. In some embodiments,the methods comprise: contacting a sample with a polynucleotide thatcorresponds to a differentially expressed gene described herein underconditions that allow hybridization; and detecting hybridization, ifany. Detection of differential hybridization, when compared to asuitable control, is an indication of the presence in the sample of apolynucleotide that is differentially expressed in a cancer cell.Appropriate controls include, for example, a sample that is known not tocontain a polynucleotide that is differentially expressed in a cancercell. Conditions that allow hybridization are known in the art, and havebeen described in more detail above.

Detection can also be accomplished by any known method, including, butnot limited to, in situ hybridization, PCR (polymerase chain reaction),RT-PCR (reverse transcription-PCR), and “Northern” or RNA blotting,arrays, microarrays, etc, or combinations of such techniques, using asuitably labeled polynucleotide. A variety of labels and labelingmethods for polynucleotides are known in the art and can be used in theassay methods of the invention. Specific hybridization can be determinedby comparison to appropriate controls.

Labeled nucleic acid probes may be used to detect expression of a genecorresponding to the provided polynucleotide, e.g. in a macroarrayformat, Northern blot, etc. The amount of hybridization can bequantitated to determine relative amounts of expression, for exampleunder a particular condition. Probes are used for in situ hybridizationto cells to detect expression. Probes can also be used in vivo fordiagnostic detection of hybridizing sequences. Probes may be labeledwith a radioactive isotope. Other types of detectable labels can be usedsuch as chromophores, fluorophores, and enzymes.

Polynucleotide arrays provide a high throughput technique that can assaya large number of polynucleotides or polypeptides in a sample. Thistechnology can be used as a tool to test for differential expression. Avariety of methods of producing arrays, as well as variations of thesemethods, are known in the art and contemplated for use in the invention.For example, arrays can be created by spotting polynucleotide probesonto a substrate (e.g., glass, nitrocellulose, etc.) in atwo-dimensional matrix or array having bound probes. The probes can bebound to the substrate by either covalent bonds or by non-specificinteractions, such as hydrophobic interactions.

Characterization of Acute Myeloid Leukemia Stem Cells

In acute myeloid leukemias, characterization of cancer stem cells allowsfor the development of new treatments that are specifically targetedagainst this critical population of cells, particularly their ability toself-renew, resulting in more effective therapies.

In human acute myeloid leukemias it is shown herein that there is asubpopulation of tumorigenic cancer cells with both self-renewal anddifferentiation capacity. These tumorigenic cells are responsible fortumor maintenance, and also give rise to large numbers of abnormallydifferentiating progeny that are not tumorigenic, thus meeting thecriteria of cancer stem cells. Tumorigenic potential is contained withina subpopulation of cancer cells differentially expressing the markers ofthe present invention.

In some embodiments of the invention, the number of AMLSC in a patientsample is determined relative to the total number of AML cancer cells,where a greater percentage of AMLSC is indicative of the potential forcontinued self-renewal of cells with the cancer phenotype. Thequantitation of AMLSC in a patient sample may be compared to a referencepopulation, e.g. a patient sample such as a blood sample, a remissionpatient sample, etc. In some embodiments, the quantitation of AMLSC isperformed during the course of treatment, where the number of AML cancercells and the percentage of such cells that are AMLSC are quantitatedbefore, during and as follow-up to a course of therapy. Desirably,therapy targeted to cancer stem cells results in a decrease in the totalnumber, and/or percentage of AMLSC in a patient sample.

In other embodiments of the invention, anti-cancer agents are targetedto AMLSC by specific binding to a marker or combination of markers ofthe present invention. In such embodiments, the anti-cancer agentsinclude antibodies and antigen-binding derivatives thereof specific fora marker or combination of markers of the present invention, which areoptionally conjugated to a cytotoxic moiety. Depletion of AMLSC isuseful in the treatment of AML. Depletion achieves a reduction incirculating AMLSC by up to about 30%, or up to about 40%, or up to about50%, or up to about 75% or more. Depletion can be achieved by using a anagent to deplete AMLSC either in vivo or ex vivo.

The AMLSC are identified by their phenotype with respect to particularmarkers, and/or by their functional phenotype. In some embodiments, theAMLSC are identified and/or isolated by binding to the cell withreagents specific for the markers of interest. The cells to be analyzedmay be viable cells, or may be fixed or embedded cells.

In some embodiments, the reagents specific for the markers of interestare antibodies, which may be directly or indirectly labeled. Suchantibodies will usually include antibodies specific for a marker orcombination of markers of the present invention.

Treatment of Cancer

The invention further provides methods for reducing growth of cancercells. The methods provide for decreasing the number of cancer cellsbearing a specific marker or combination of markers, as provided herein,decreasing expression of a gene that is differentially expressed in acancer cell, or decreasing the level of and/or decreasing an activity ofa cancer-associated polypeptide. In general, the methods comprisecontacting a cancer cell with a binding agent, e.g. an antibody orligand specific for a marker or combination of markers provided herein.

“Reducing growth of cancer cells” includes, but is not limited to,reducing proliferation of cancer cells, and reducing the incidence of anon-cancerous cell becoming a cancerous cell. Whether a reduction incancer cell growth has been achieved can be readily determined using anyknown assay, including, but not limited to, [³H]-thymidineincorporation; counting cell number over a period of time; detectingand/or measuring a marker associated with AML, etc.

The present invention provides methods for treating cancer, generallycomprising administering to an individual in need thereof a substancethat reduces cancer cell growth, in an amount sufficient to reducecancer cell growth and treat the cancer. Whether a substance, or aspecific amount of the substance, is effective in treating cancer can beassessed using any of a variety of known diagnostic assays for cancer,including, but not limited to biopsy, contrast radiographic studies, CATscan, and detection of a tumor marker associated with cancer in theblood of the individual. The substance can be administered systemicallyor locally, usually systemically.

A substance, e.g. a chemotherapeutic drug that reduces cancer cellgrowth, can be targeted to a cancer cell. Thus, in some embodiments, theinvention provides a method of delivering a drug to a cancer cell,comprising administering a drug-antibody complex to a subject, whereinthe antibody is specific for a cancer-associated polypeptide, and thedrug is one that reduces cancer cell growth, a variety of which areknown in the art. Targeting can be accomplished by coupling (e.g.,linking, directly or via a linker molecule, either covalently ornon-covalently, so as to form a drug-antibody complex) a drug to anantibody specific for a cancer-associated polypeptide. Methods ofcoupling a drug to an antibody are well known in the art and need not beelaborated upon herein.

Staging and Diagnosis

Acute myeloid leukemias are staged by analysis of the presence of cancerstem cells. Staging is useful for prognosis and treatment. In oneembodiment of the invention, a sample from an acute myeloid leukemiapatient is stained with reagents specific for a marker or combination ofmarkers of the present invention. The analysis of staining patternsprovides the relative distribution of AMLSC, which distribution predictsthe stage of leukemia. In some embodiments, the sample is analyzed byhistochemistry, including immunohistochemistry, in situ hybridization,and the like, for the presence of CD34⁺CD38⁻ cells that express a markeror combination of markers of the present invention. The presence of suchcells indicates the presence of AMLSC.

In one embodiment, the patient sample is compared to a control, or astandard test value. In another embodiment, the patient sample iscompared to a pre-leukemia sample, or to one or more time points throughthe course of the disease.

Samples, including tissue sections, slides, etc. containing an acutemyeloid leukemia tissue, are stained with reagents specific for markersthat indicate the presence of cancer stem cells. Samples may be frozen,embedded, present in a tissue microarray, and the like. The reagents,e.g. antibodies, polynucleotide probes, etc. may be detectably labeled,or may be indirectly labeled in the staining procedure. The dataprovided herein demonstrate that the number and distribution ofprogenitor cells is diagnostic of the stage of the leukemia.

The information thus derived is useful in prognosis and diagnosis,including susceptibility to acceleration of disease, status of adiseased state and response to changes in the environment, such as thepassage of time, treatment with drugs or other modalities. The cells canalso be classified as to their ability to respond to therapeutic agentsand treatments, isolated for research purposes, screened for geneexpression, and the like. The clinical samples can be furthercharacterized by genetic analysis, proteomics, cell surface staining, orother means, in order to determine the presence of markers that areuseful in classification. For example, genetic abnormalities can becausative of disease susceptibility or drug responsiveness, or can belinked to such phenotypes.

Differential Cell Analysis

The presence of AMLSC in a patient sample can be indicative of the stageof the leukemia. In addition, detection of AMLSC can be used to monitorresponse to therapy and to aid in prognosis. The presence of AMLSC canbe determined by quantitating the cells having the phenotype of the stemcell. In addition to cell surface phenotyping, it may be useful toquantitate the cells in a sample that have a “stem cell” character,which may be determined by functional criteria, such as the ability toself-renew, to give rise to tumors in vivo, e.g. in a xenograft model,and the like.

Clinical samples for use in the methods of the invention may be obtainedfrom a variety of sources, particularly blood, although in someinstances samples such as bone marrow, lymph, cerebrospinal fluid,synovial fluid, and the like may be used. Such samples can be separatedby centrifugation, elutriation, density gradient separation, apheresis,affinity selection, panning, FACS, centrifugation with Hypaque, etc.prior to analysis, and usually a mononuclear fraction (PBMC) will beused. Once a sample is obtained, it can be used directly, frozen, ormaintained in appropriate culture medium for short periods of time.Various media can be employed to maintain cells. The samples may beobtained by any convenient procedure, such as the drawing of blood,venipuncture, biopsy, or the like. Usually a sample will comprise atleast about 10² cells, more usually at least about 10³ cells, andpreferable 10⁴, 10⁵ or more cells. Typically the samples will be fromhuman patients, although animal models may find use, e.g. equine,bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster,primate, etc.

An appropriate solution may be used for dispersion or suspension of thecell sample. Such solution will generally be a balanced salt solution,e.g. normal saline, PBS, Hank's balanced salt solution, etc.,conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, generally from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc.

Analysis of the cell staining will use conventional methods. Techniquesproviding accurate enumeration include fluorescence activated cellsorters, which can have varying degrees of sophistication, such asmultiple color channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. The cells may be selected againstdead cells by employing dyes associated with dead cells (e.g. propidiumiodide).

The affinity reagents may be specific receptors or ligands for the cellsurface molecules indicated above. In addition to antibody reagents,peptide-MHC antigen and T cell receptor pairs may be used; peptideligands and receptors; effector and receptor molecules, and the like.Antibodies and T cell receptors may be monoclonal or polyclonal, and maybe produced by transgenic animals, immunized animals, immortalized humanor animal B-cells, cells transfected with DNA vectors encoding theantibody or T cell receptor, etc. The details of the preparation ofantibodies and their suitability for use as specific binding members arewell-known to those skilled in the art.

Of particular interest is the use of antibodies as affinity reagents.Conveniently, these antibodies are conjugated with a label for use inseparation. Labels include magnetic beads, which allow for directseparation, biotin, which can be removed with avidin or streptavidinbound to a support, fluorochromes, which can be used with a fluorescenceactivated cell sorter, or the like, to allow for ease of separation ofthe particular cell type. Fluorochromes that find use includephycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluoresceinand Texas red. Frequently each antibody is labeled with a differentfluorochrome, to permit independent sorting for each marker.

The antibodies are added to a suspension of cells, and incubated for aperiod of time sufficient to bind the available cell surface antigens.The incubation will usually be at least about 5 minutes and usually lessthan about 30 minutes. It is desirable to have a sufficientconcentration of antibodies in the reaction mixture, such that theefficiency of the separation is not limited by lack of antibody. Theappropriate concentration is determined by titration. The medium inwhich the cells are separated will be any medium that maintains theviability of the cells. A preferred medium is phosphate buffered salinecontaining from 0.1 to 0.5% BSA. Various media are commerciallyavailable and may be used according to the nature of the cells,including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic SaltSolution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI,Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplemented withfetal calf serum, BSA, HSA, etc.

The labeled cells are then quantitated as to the expression of cellsurface markers as previously described.

The comparison of a differential progenitor analysis obtained from apatient sample, and a reference differential progenitor analysis isaccomplished by the use of suitable deduction protocols, AI systems,statistical comparisons, etc. A comparison with a reference differentialprogenitor analysis from normal cells, cells from similarly diseasedtissue, and the like, can provide an indication of the disease staging.A database of reference differential progenitor analyses can becompiled. An analysis of particular interest tracks a patient, e.g. inthe chronic and pre-leukemic stages of disease, such that accelerationof disease is observed at an early stage. The methods of the inventionprovide detection of acceleration prior to onset of clinical symptoms,and therefore allow early therapeutic intervention, e.g. initiation ofchemotherapy, increase of chemotherapy dose, changing selection ofchemotherapeutic drug, and the like.

AMLSC Compositions

AMLSC may be separated from a complex mixture of cells by techniquesthat enrich for cells that differentially express a marker orcombination of markers of the present invention. For isolation of cellsfrom tissue, an appropriate solution may be used for dispersion orsuspension. Such solution will generally be a balanced salt solution,e.g. normal saline, PBS, Hank's balanced salt solution, etc.,conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, generally from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc.

The separated cells may be collected in any appropriate medium thatmaintains the viability of the cells, usually having a cushion of serumat the bottom of the collection tube. Various media are commerciallyavailable and may be used according to the nature of the cells,including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequentlysupplemented with fetal calf serum.

Compositions highly enriched for AMLSC are achieved in this manner. Thesubject population may be at or about 50% or more of the cellcomposition, and preferably be at or about 75% or more of the cellcomposition, and may be 90% or more. The desired cells are identified bytheir surface phenotype, by the ability to self-renew, ability to formtumors, etc. The enriched cell population may be used immediately, ormay be frozen at liquid nitrogen temperatures and stored for longperiods of time, being thawed and capable of being reused. The cells maybe stored in 10% DMSO, 90% FCS medium. The population of cells enrichedfor AMLSC may be used in a variety of screening assays and cultures, asdescribed below.

The enriched AMLSC population may be grown in vitro under variousculture conditions. Culture medium may be liquid or semi-solid, e.g.containing agar, methylcellulose, etc. The cell population may beconveniently suspended in an appropriate nutrient medium, such asIscove's modified DMEM or RPMI-1640, normally supplemented with fetalcalf serum (about 5-10%), L-glutamine, a thiol, particularly2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin.

The culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors. A wide variety of growth factors may be used inculturing the cells, e.g. LIF, steel factor (c-kit ligand), EGF,insulin, IGF, Flk-2 ligand, IL-11, IL-3, GM-CSF, erythropoietin,thrombopoietin, etc

In addition to, or instead of growth factors, the subject cells may begrown in a co-culture with fibroblasts, stromal or other feeder layercells. Stromal cells suitable for use in the growth of hematopoieticcells are known in the art. These include bone marrow stroma as used in“Whitlock-Witte” (Whitlock et al. [1985] Annu Rev Immunol 3:213-235) or“Dexter” culture conditions (Dexter et al. [1977] J Exp Med145:1612-1616); and heterogeneous thymic stromal cells.

Screening Assays

AMLSC expressing a marker or combination of markers of the presentinvention are also useful for in vitro assays and screening to detectfactors and chemotherapeutic agents that are active on cancer stemcells. Of particular interest are screening assays for agents that areactive on human cells. A wide variety of assays may be used for thispurpose, including immunoassays for protein binding; determination ofcell growth, differentiation and functional activity; production offactors; and the like. In other embodiments, isolated polypeptidescorresponding to a marker or combination of markers of the presentinvention are useful in drug screening assays.

In screening assays for biologically active agents, anti-proliferativedrugs, etc. the marker or AMLSC composition is contacted with the agentof interest, and the effect of the agent assessed by monitoring outputparameters on cells, such as expression of markers, cell viability, andthe like; or binding efficacy or effect on enzymatic or receptoractivity for polypeptides. The cells may be freshly isolated, cultured,genetically altered, and the like. The cells may be environmentallyinduced variants of clonal cultures: e.g. split into independentcultures and grown under distinct conditions, for example with orwithout drugs; in the presence or absence of cytokines or combinationsthereof. The manner in which cells respond to an agent, particularly apharmacologic agent, including the timing of responses, is an importantreflection of the physiologic state of the cell.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can be any cell component or cell product including cellsurface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Characteristically a range of parameter readoutvalues will be obtained for each parameter from a multiplicity of thesame assays. Variability is expected and a range of values for each ofthe set of test parameters will be obtained using standard statisticalmethods with a common statistical method used to provide single values.

Agents of interest for screening include known and unknown compoundsthat encompass numerous chemical classes, primarily organic molecules,which may include organometallic molecules, inorganic molecules, geneticsequences, etc. An important aspect of the invention is to evaluatecandidate drugs, including toxicity testing; and the like.

In addition to complex biological agents candidate agents includeorganic molecules comprising functional groups necessary for structuralinteractions, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, frequently atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomolecules,including peptides, polynucleotides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Included are pharmacologically active drugs, genetically activemolecules, etc. Compounds of interest include chemotherapeutic agents,hormones or hormone antagonists, etc. Exemplary of pharmaceutical agentssuitable for this invention are those described in, “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Water, Salts and Ions; DrugsAffecting Renal Function and Electrolyte Metabolism; Drugs AffectingGastrointestinal Function; Chemotherapy of Microbial Diseases;Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Formingorgans; Hormones and Hormone Antagonists; Vitamins, Dermatology; andToxicology, all incorporated herein by reference. Also included aretoxins, and biological and chemical warfare agents, for example seeSomani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, NewYork, 1992).

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. Of interest arecomplex mixtures of naturally occurring compounds derived from naturalsources such as plants. While many samples will comprise compounds insolution, solid samples that can be dissolved in a suitable solvent mayalso be assayed. Samples of interest include environmental samples, e.g.ground water, sea water, mining waste, etc.; biological samples, e.g.lysates prepared from crops, tissue samples, etc.; manufacturingsamples, e.g. time course during preparation of pharmaceuticals; as wellas libraries of compounds prepared for analysis; and the like. Samplesof interest include compounds being assessed for potential therapeuticvalue, i.e. drug candidates.

The term “samples” also includes the fluids described above to whichadditional components have been added, for example components thataffect the ionic strength, pH, total protein concentration, etc. Inaddition, the samples may be treated to achieve at least partialfractionation or concentration. Biological samples may be stored if careis taken to reduce degradation of the compound, e.g. under nitrogen,frozen, or a combination thereof. The volume of sample used issufficient to allow for measurable detection, usually from about 0.1 to1 ml of a biological sample is sufficient.

Compounds, including candidate agents, are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds, including biomolecules,including expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

Agents are screened for biological activity by adding the agent to atleast one and usually a plurality of cell samples, usually inconjunction with cells lacking the agent. The change in parameters inresponse to the agent is measured, and the result evaluated bycomparison to reference cultures, e.g. in the presence and absence ofthe agent, obtained with other agents, etc.

The agents are conveniently added in solution, or readily soluble form,to the medium of cells in culture. The agents may be added in aflow-through system, as a stream, intermittent or continuous, oralternatively, adding a bolus of the compound, singly or incrementally,to an otherwise static solution. In a flow-through system, two fluidsare used, where one is a physiologically neutral solution, and the otheris the same solution with the test compound added. The first fluid ispassed over the cells, followed by the second. In a single solutionmethod, a bolus of the test compound is added to the volume of mediumsurrounding the cells. The overall concentrations of the components ofthe culture medium should not change significantly with the addition ofthe bolus, or between the two solutions in a flow through method.

Preferred agent formulations do not include additional components, suchas preservatives, that may have a significant effect on the overallformulation. Thus preferred formulations consist essentially of abiologically active compound and a physiologically acceptable carrier,e.g. water, ethanol, DMSO, etc. However, if a compound is liquid withouta solvent, the formulation may consist essentially of the compounditself.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype.

Various methods can be utilized for quantifying the presence of theselected markers. For measuring the amount of a molecule that ispresent, a convenient method is to label a molecule with a detectablemoiety, which may be fluorescent, luminescent, radioactive,enzymatically active, etc., particularly a molecule specific for bindingto the parameter with high affinity. Fluorescent moieties are readilyavailable for labeling virtually any biomolecule, structure, or celltype. Immunofluorescent moieties can be directed to bind not only tospecific proteins but also specific conformations, cleavage products, orsite modifications like phosphorylation. Individual peptides andproteins can be engineered to autofluoresce, e.g. by expressing them asgreen fluorescent protein chimeras inside cells (for a review see Joneset al. (1999) Trends Biotechnol. 17(12):477-81). Thus, antibodies can begenetically modified to provide a fluorescent dye as part of theirstructure. Depending upon the label chosen, parameters may be measuredusing other than fluorescent labels, using such immunoassay techniquesas radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA),homogeneous enzyme immunoassays, and related non-enzymatic techniques.The quantitation of nucleic acids, especially messenger RNAs, is also ofinterest as a parameter. These can be measured by hybridizationtechniques that depend on the sequence of nucleic acid nucleotides.Techniques include polymerase chain reaction methods as well as genearray techniques. See Current Protocols in Molecular Biology, Ausubel etal., eds, John Wiley & Sons, New York, N.Y., 2000; Freeman et al. (1999)Biotechniques 26(1):112-225; Kawamoto et al. (1999) Genome Res9(12):1305-12; and Chen et al. (1998) Genomics 51(3):313-24, forexamples.

Depletion of AMLSC

Depletion of AMLSC is useful in the treatment of AML. Depletion can beachieved by several methods. Depletion is defined as a reduction in thetarget population by up to about 30%, or up to about 40%, or up to about50%, or up to about 75% or more. An effective depletion is usuallydetermined by the sensitivity of the particular disease condition to thelevels of the target population. Thus in the treatment of certainconditions a depletion of even about 20% could be beneficial.

A marker-specific agent that specifically depletes the targeted AMLSC isused to contact the patient blood in vitro or in vivo, wherein after thecontacting step, there is a reduction in the number of viable AMLSC inthe targeted population. An exemplary agent for such purposes is anantibody that specifically binds to a marker or combination of markersof the present invention on the surface of the targeted AMLSC. Aneffective dose of antibodies for such a purpose is sufficient todecrease the targeted population to the desired level, for example asdescribed above. Antibodies for such purposes may have low antigenicityin humans or may be humanized antibodies.

In one embodiment of the invention, antibodies for depleting targetpopulation are added to patient blood in vivo. In another embodiment,the antibodies are added to the patient blood ex vivo. Beads coated withthe antibody of interest can be added to the blood, target cells boundto these beads can then be removed from the blood using procedurescommon in the art. In one embodiment the beads are magnetic and areremoved using a magnet. Alternatively, when the antibody isbiotinylated, it is also possible to indirectly immobilize the antibodyonto a solid phase which has adsorbed avidin, streptavidin, or the like.The solid phase, usually agarose or sepharose beads are separated fromthe blood by brief centrifugation. Multiple methods for taggingantibodies and removing such antibodies and any cells bound to theantibodies are routine in the art. Once the desired degree of depletionhas been achieved, the blood is returned to the patient. Depletion oftarget cells ex vivo decreases the side effects such as infusionreactions associated with the intravenous administration. An additionaladvantage is that the repertoire of available antibodies is expandedsignificantly as this procedure does not have to be limited toantibodies with low antigenicity in humans or humanized antibodies.

Kits may be provided, where the kit will comprise staining reagents thatare sufficient to differentially identify the AMLSC described herein. Acombination of interest may include one or more reagents specific for amarker or combination of markers of the present invention, and mayfurther include antibodies specific for CD96, CD34, and CD38. Thestaining reagents are preferably antibodies, and may be detectablylabeled. Kits may also include tubes, buffers, etc., and instructionsfor use.

Each publication cited in this specification is hereby incorporated byreference in its entirety for all purposes.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

EXPERIMENTAL Example 1 Identification of a Hierarchy of MultipotentHematopoietic Progenitors in Human Cord Blood

Mouse hematopoiesis is initiated by long-term hematopoietic stem cells(HSC) that differentiate into a series of multipotent progenitors thatexhibit progressively diminished self-renewal ability. In humanhematopoiesis, populations enriched for HSC activity have beenidentified, as have downstream lineage-committed progenitors, butmultipotent progenitor activity has not been uniquely isolated. Previousreports indicate that human HSC are enriched in Lin−CD34+CD38− cordblood and bone marrow and express CD90. We demonstrate that theLin−CD34+CD38− fraction of cord blood and bone marrow can be subdividedinto three subpopulations: CD90+CD45RA−, CD90−CD45RA−, and CD90−CD45RA+.Utilizing in vivo transplantation studies and complementary in vitroassays, we demonstrate that the Lin−CD34+CD38−CD90+CD45RA− cord bloodfraction contains HSC, and isolate this activity to as few as 10purified cells. Furthermore, we report the first prospective isolationof a population of candidate human multipotent progenitors,Lin−CD34+CD38−CD90−CD45RA− cord blood.

Identification of CD90/CD45RA Subpopulations of Lin−CD34+CD38− HumanBone Marrow and Cord Blood. Data from multiple investigators indicatethat human HSC activity resides in the CD90+ fraction ofLin−CD34+CD38−/lo cells. Using the marker CD45RA, three subpopulationsof Lin−CD34+CD38− bone marrow and cord blood were identified: (1)CD90+CD45RA−, (2) CD90−CD45RA−, and (3) CD90−CD45RA+(FIG. 1). In thebone marrow these population comprised 30.3±18.9% (CD90+CD45RA−),37.7±14.1% (CD90−CD45RA−), and 24.7±11.8% (CD90−CD45RA) ofLin−CD34+CD38− cells (n=10). In cord blood, these fractions constituted25.2±10.3% (CD90+CD45RA−), 49.8±11.4% (CD90−CD45RA−), and 18.4±8.4%(CD90−CD45RA+) of Lin−CD34+CD38− cells (n=22). All three subpopulationswere isolated to >95% purity from cord blood and bone marrow by FACS.

Methylcellulose Colony Formation and Replating of CD90/CD45RASubpopulations. In order to assess the lineage potential of theCD90/CD45RA subpopulations, each was assayed for in vitro colonyformation in methylcellulose. Single cells of each population weresorted into individual wells of a 96-well plate containing completemethylcellulose. In all cases, only a single colony was detected.CD90+CD45RA− cells formed all types of myeloid colonies, as didCD90−CD45RA− cells (FIG. 2A). No differences were detected in platingefficiency or colony subtype distribution between these twosubpopulations; however, the CD90+CD45RA− colonies were generally muchlarger and faster growing. CD90−CD45RA+ cells formed very few colonies,suggesting that these cells possess limited myeloid differentiationpotential.

All colonies derived from individual cells were then harvested,dissociated, and plated in complete methylcellulose in order todetermine replating efficiency, an in vitro surrogate for self-renewal(FIG. 2B). 70% of colonies derived from CD90+CD45RA− cells were able toform new colonies (in most cases, hundreds) upon replating. 33% ofcolonies derived from CD90−CD45RA− cells were able to form new colonies(in most cases, fewer than 50). None of the colonies derived fromCD90+CD45RA− cells were able to form new colonies; however, very fewcolonies formed in the first plating (n=6). Thus, the CD90−CD45RA−subpopulation is able to form all myeloid cells, but has reducedcapacity for self-renewal compared to the CD90+CD45RA− subpopulation,which is presumed to contain HSC.

In Vitro Proliferation and Differentiation Identifies a Hierarchy Amongthe CD90/CD45RA Subpopulations. The CD90/CD45RA subpopulations were alsoassayed for in vitro proliferation in serum-free liquid culture. Singlecells of each population were sorted into individual wells of a 96-wellplate containing serum-free media supplemented with Flt-3 ligand, SCF,TPO, IL-3, and IL-6 and cultured for 2 weeks. At the end of the cultureperiod, live cells were counted. CD90+CD45RA− cells proliferatedextensively with a mean recovery of 345,000 cells; CD90−CD45RA− cellsproliferated to a lesser degree with a mean recovery of 67,500 cells;CD90−CD45RA+ cells proliferated poorly with few live cells recovered(FIG. 2C). These results support the observed differences inmethylcellulose colony size and suggest that the CD90− subpopulationsare less primitive than the CD90+ subpopulation presumed to contain HSC.

In order to examine the potential hierarchical relationships between theCD90/CD45RA subpopulations within the Lin−CD34+CD38− fraction, eachpopulation was sorted in bulk into serum-free media supplemented withcytokines as described above. After four days in culture, the cells werere-analyzed for expression of CD90 and CD45RA (FIG. 2D). WhileCD90+CD45RA− cells gave rise to all three subpopulations, CD90−CD45RA−cells gave rise to both CD90− subpopulations, but not CD90+ cells.CD90−CD45RA+ cells gave rise principally to itself only. Together, thesedata establish an in vitro differentiation hierarchy in whichCD90+CD45RA− cells give rise to CD90−CD45RA− cells, which in turn giverise to CD90−CD45RA+ cells.

Long-Term In Vivo Multipotent Human Hematopoiesis with Transplantationof as few as 10 Lin−CD34+CD38−CD90+CD45RA− Cord Blood Cells. NewbornNOD/SCID/IL-2^(γ)-null (NOG) mice were used in xenotransplantationassays to determine the differentiation potential and self-renewalability of the CD90/CD45RA subpopulations. Transplantation of 100purified CD90+CD45RA− cells resulted in circulating human CD45+hematopoietic cells at 12 weeks, including both CD13+ myeloid cells, andCD19+ B cells, but not CD3+ T cells (FIG. 3A). Several of these micewere followed beyond 12 weeks (maximum 30 weeks), and all continued tohave detectable human myeloid cells at similar levels in the peripheralblood (data not shown), indicating continued human engraftment. In orderto isolate this activity to as few cells as possible, 10 purifiedCD90+CD45RA− cells were transplanted. At 12 weeks, few circulating humanCD45+ cells were detected, and no CD13+ myeloid cells were present (FIG.3A); however, analysis of the bone marrow showed significant humanengraftment with both myeloid and lymphoid cells (FIG. 3A). Successfullong-term human engraftment was observed in 3 out of 10 micetransplanted with 10 CD90+CD45RA− cells, with the 3 successfulengraftments coming from independent cord blood samples.

Both CD13+ myeloid cells and CD19+ B cells were detected in the bloodand bone marrow of engrafted mice, indicating that CD90+CD45RA− cellspossess lymphoid and myeloid potential, and are likely multipotent.Analysis of the spleen from engrafted mice identified human CD45+CD3+ Tcells, and staining of the bone marrow with glycophorin-A and CD61/41identified human erythroid cells and platelets. To confirm this flowcytometry-derived lineage analysis, human CD45+ cells from bothperipheral blood and bone marrow were FACS-purified and cytospinpreparations were stained with Wright-Giemsa. These stains confirmed thepresence of mature human lymphocytes, neutrophils, and monocytes in theperipheral blood (FIG. 3B, panels 1-3). In the bone marrow, bothlymphocytes and maturing myeloid cells were readily detected (FIG. 3B,panel 4). Collectively, these data indicate thatLin−CD34+CD38−CD90+CD45RA− cord blood cells are capable of establishinglong-term in vivo multipotent human hematopoiesis and that this activitycan be isolated to as few as 10 cells.

Long-Term In Vivo Multipotent Human Hematopoiesis RequiresTransplantation of More CD90−CD45RA− Cells Than CD90+CD45RA− Cells. Allthree CD90/CD45RA subpopulations of cord blood were purified by FACS andtransplanted into NOG mice. Transplantation of both the CD90+CD45RA− andthe CD90−CD45RA− subpopulations resulted in detectable human myeloid andB lymphoid cells in the peripheral blood 12 weeks after transplantation(FIG. 4A). No human cells were detected in the peripheral blood of micetransplanted with the CD90−CD45RA+ subpopulation, even at time points asearly as 4 weeks after transplantation (FIG. 4A). Multipletransplantation experiments were conducted with independent cord bloodsamples resulting in transplantation of between 100 and 4440 cells ofeach population (FIG. 4B). The cumulative data from these experimentsshowed that 12 of 13 mice (92%) transplanted with CD90+CD45RA− cells,and 15 of 17 mice (88%) transplanted with CD90−CD45RA− cells containedhuman CD45+ cells in the peripheral blood at least 12 weeks aftertransplantation (FIG. 4B). In the engrafted mice, the average humanCD45+ chimerism was 3.7% for the CD90+CD45RA− transplants compared to4.0% for the CD90−CD45RA− transplants; the percentage of myeloid cellsamong human CD45+ cells was 6.7% for the CD90+CD45RA− transplantscompared to 7.1% for the CD90−CD45RA− transplants (FIG. 4B). Becausedifferent cell numbers were used in each of these transplantationexperiments, the engraftment per 100 transplanted cells was determined.The CD90+CD45RA− engrafted mice averaged 7 fold greater human chimerismand 7 fold greater human myeloid cells than the CD90−CD45RA− engraftedmice (FIG. 4C). The difference in human chimerism was statisticallysignificant with p=0.02, while the difference in human myeloid cellsapproached statistical significance with p=0.08.

Analysis of the bone marrow of transplanted mice revealed human myeloidand B cells 12 weeks after transplantation of CD90+CD45RA− andCD90−CD45RA− cells (FIG. 5A). No human cells were detected in the bonemarrow of mice transplanted with the CD90−CD45RA+ population. Cumulativedata showed that 8 of 8 mice (100%) transplanted with CD90+CD45RA−cells, and 8 of 9 mice (89%) transplanted with CD90−CD45RA− cellscontained human CD45+ cells in the bone marrow at least 12 weeks aftertransplantation (FIG. 5B). In the engrafted mice, the average humanchimerism was 19.9% for the CD90+CD45RA− transplants compared to 4.4%for the CD90−CD45RA− transplants; the percent myeloid of total humanCD45+ cells was 24.3% for the CD90+CD45RA− transplants compared to 26.6%for the CD90−CD45RA− transplants (FIG. 5B). In order to normalize forcell number, bone marrow engraftment per 100 transplanted cells wasdetermined. The CD90+CD45RA− engrafted mice averaged 9 fold greaterhuman chimerism and 9 fold greater human myeloid cells than theCD90−CD45RA− engrafted mice (FIG. 5C). The difference in human chimerismwas statistically significant with p=0.001, while the difference inhuman myeloid cells approached statistical significance with p=0.07.

Analysis of the spleens of CD90+CD45RA− and CD90−CD45RA− transplantedmice identified human CD45+ cells consisting of rare myeloid cells,numerous B cells, and occasional T cells. No human cells were detectedin the spleens of mice transplanted with CD90−CD45RA+ cells. Significantnumbers of T cells were detected in only a subset of mice transplantedwith either engrafting population (Supplementary FIG. 2). Determiningsplenic engraftment per 100 transplanted cells revealed thatCD90+CD45RA− engrafted mice averaged 7 fold greater human chimerism thanthe CD90−CD45RA− engrafted mice, and this difference was statisticallysignificant; however, no statistically significant difference wasdetected in the T cell percentage. Finally, bone marrow from miceengrafted with CD90−CD45RA− cells was found to contain GPA-positivehuman erythroid cells and CD61/CD41-positive human platelets (data notshown), indicating that these cells are multipotent.

To investigate the minimum number of cells required for successfulengraftment, 50 or 70 CD90−CD45RA− or CD90+CD45RA− cells were doubleFACS-purified from 2 independent cord blood samples and transplantedinto NOG mice. At least 11 weeks later, bone marrow was analyzed forhuman engraftment. All mice (n=4) transplanted with CD90+CD45RA− cellscontained human myeloid and B cells, while no mice (n=5) transplantedwith CD90−CD45RA− cells did (FIG. 5D). This difference was statisticallysignificant with p=0.008. One of the CD90−CD45RA− transplanted mice didcontain a small population (0.2%) of human T cells, indicating that itmust have engrafted at an early time point. To directly investigateearly engraftment, 50 double FACS-purified cells were transplanted intoNOG newborn mice. 4 weeks later, bone marrow of all transplanted mice,both CD90+CD45RA− (n=2) and CD90−CD45RA− (n=2), contained human myeloidand B cells.

In summary, both the CD90+CD45RA− and CD90−CD45RA− subpopulations arecapable of establishing long-term in vivo multipotent humanhematopoiesis, with similar percentages of myeloid and lymphoid cellproduction. However, the CD90−CD45RA− cells have a lower engraftmentcapacity as they require transplantation of more cells for long-termengraftment and generate fewer human cells per cell-equivalenttransplant.

In Vivo Analysis of Human CD34+ Cells Identifies a Hierarchy Among theCD90/CD45RA Subpopulations. In order to assess the hierarchicalrelationships among the CD90/CD45RA subpopulations in vivo, human CD34+progenitor cells from the bone marrows of engrafted mice were analyzed12 weeks after transplantation. In mice transplanted with CD90+CD45RA−cells the bone marrow contained, on average, 6.4% human CD34+ cellscompared to 1.3% human CD34+ cells in mice transplanted withCD90−CD45RA− cells (FIG. 6A). CD34+ engraftment per 100 transplantedcells averaged 8 fold greater in CD90+CD45RA− engrafted mice thanCD90−CD45RA− engrafted mice, and this difference was statisticallysignificant (FIG. 6B). This 8 fold statistically significant differencewas also present when comparing the percentage of Lin−CD34+ cells intotal bone marrow (data not shown). Within the engrafted Lin−CD34+fraction, there were no differences between CD90+CD45RA− andCD90−CD45RA− engrafted mice with respect to the percentages ofCD34+CD38+ or CD34+CD38− cells (FIG. 6A). Thus, CD90−CD45RA− cellsappear to be able to generate progenitor cells in similar proportions toCD90+CD45RA− cells, but are less efficient in doing so in vivo.

Human Lin−CD34+CD38− cells in the bone marrow of engrafted mice werealso analyzed for the expression of CD90 and CD45RA. All threeCD90/CD45RA subpopulations were detected in the bone marrow of micetransplanted with CD90+CD45RA− cells; however, only the two CD90−subpopulations were detected in mice transplanted with CD90−CD45RA−cells (FIG. 6C, D). In some mice transplanted with CD90−CD45RA− cells,only CD90−CD45RA+ cells were detected. Together, these data establish anin vivo differentiation hierarchy among Lin−CD34+CD38− cord blood cellsproceeding from CD90+CD45RA− to CD90−CD45RA− to CD90−CD45RA+ cells.

Enrichment of Secondary Transplant Ability in the CD90+CD45RA−Subpopulation. Self-renewal is the key property distinguishing HSC fromMPP, and is defined by the ability to sustain long-term hematopoiesisand generate successful secondary transplants. Since both theCD90+CD45RA− and CD90−CD45RA− subpopulations demonstrated the ability toestablish long-term in vivo multipotent hematopoiesis, we next assessedtheir ability to generate successful secondary transplants. Human CD34+cells were purified from whole bone marrow of primary engrafted mice andequal numbers of CD34+ cells were transplanted into NOG newborn mice.Bone marrows from these secondary recipients were analyzed at least 10weeks after transplantation. In one experiment, 130,000 human CD34+cells were transplanted into secondary recipients. Human CD45+ cells andhuman myeloid cells were detected in 8 of 8 mice (100%) from CD90+primary transplants, but only 2 of 5 mice (40%) from CD90− primarytransplants (FIG. 7A,B). In a second experiment, 70,000 human CD34+cells were transplanted into secondary recipients. Human CD45+ cells andhuman myeloid cells were detected in 4 of 4 mice (100%) from CD90+primary transplants, but only 1 of 3 mice (40%) from CD90− primarytransplants (FIG. 7A,C). This difference, 12 of 12 (100%) for CD90+versus 3 of 8 (37.5%) for CD90−, was statistically significant withp=0.004. These data indicate that the CD90+CD45RA− subpopulation isenriched for the ability to generate successful secondary transplantscompared to the CD90−CD45RA− subpopulation.

HSC are defined by two key functional properties: (1) multipotency,defined as the ability to form all differentiated blood cells, and (2)long-term self-renewal, defined as the ability to give rise to progenyidentical to the parent through cell division. It is this property ofself-renewal that distinguishes HSC from multipotent progenitors, and isexperimentally demonstrated through the ability to generate successfulsecondary transplants. We show here that both CD90+CD45RA− andCD90−CD45RA− cord blood cells are able to establish long-termmultipotent hematopoiesis in vivo, and that the CD90+CD45RA−subpopulation is enriched for the ability to generate successfulsecondary transplants. We conclude that the CD90+CD45RA− subpopulationcontains HSC, while the CD90−CD45RA− subpopulation contains candidatemultipotent progenitors. This represents the first identification andprospective isolation of a population of candidate human multipotentprogenitors.

Both the CD90+CD45RA− and CD90−CD45RA− cells are able to establishmultipotent long-term human hematopoiesis in vivo; however, theCD90−CD45RA− subpopulation requires more cells to accomplish this asindicated by the failure of 50 and 70 cell transplants to long-termengraft, unlike the CD90+CD45RA− subpopulation which engrafts long-termwith as few as 10 cells. The fact that 50 CD90−CD45RA− cells showmyeloid and B lymphoid engraftment at 4 weeks, but not 12 weeks,indicates that this fraction contains non-HSC multipotent cells. Thus,we have demonstrated that CD90−CD45RA− cells are multipotent, exhibit areduced and incomplete capacity for self-renewal, and lie downstream ofCD90+CD45RA− cells in the hematopoietic hierarchy. We conclude thatCD90−CD45RA− cells are a multipotent hematopoietic progenitor.

Hematopoiesis proceeds through an organized hierarchy in which lineagepotential becomes increasingly restricted and a given population canonly give rise to downstream populations. We investigated thehierarchical relationships between the CD90/CD45RA subpopulations ofLin−CD34+CD38− cord blood and found that both in vitro and in vivo, theCD90+CD45RA− population gives rise to itself and both the CD90−CD45RA−and CD90−CD45RA+ subpopulations. CD90−CD45RA− cells do not give rise toCD90+ cells, but can form both CD90− subpopulations. In vitro theCD90−CD45RA+ cells give rise principally to itself only. These resultsestablish a hierarchy among Lin−CD34+CD38− cord blood cells in whichCD90+CD45RA− cells are upstream of CD90−CD45RA− cells, which areupstream of CD90−CD45RA+ cells.

CD90 and CD45RA identify a third population within the Lin−CD34+CD38−fraction of cord blood and bone marrow, the CD90−CD45RA+ subpopulation.Transplantation with up to 900 of these cells does not result in anycirculating human hematopoietic cells at 4 weeks after transplantation,and there are no detectable human cells in the bone marrow at 12 weeks.Furthermore, these cells have extremely poor methylcellulose colonyforming ability, yielding only 6 colonies from 180 plated cells,suggesting that they possess limited myeloid differentiation potential.These cells also did not proliferate in liquid culture under conditionsable to promote the growth of the other two subpopulations. It isinteresting to note, that cells with this immunophenotype can be foundwithin the bone marrow of mice engrafted with either the CD90+CD45RA− orCD90−CD45RA− subpopulation. Thus, it is likely that they contribute toongoing human hematopoiesis, but at this time cannot be placed withinthe hematopoietic hierarchy.

Numerous xenotransplantation experiments have reported the successfulenrichment of human HSC activity in Lin−CD34+CD38−/lo fractions of humanhematopoietic progenitors through the demonstration of long-termmultipotent engraftment and successful secondary transplantation. Inpublished reports, successful secondary engraftment has required primarytransplantation of large numbers of cells, minimally thousands andusually many more. We report here long-term in vivo human engraftmentand successful secondary engraftment with transplantation of 500purified Lin−CD34+CD38−CD90+CD45RA− cord blood cells. The majordifferences between our results and previous reports are: (1) the use ofthe NOG newborn mice, which appear to be well-suited for humanhematopoietic engraftment, and (2) the combination ofLin−CD34+CD38−CD90+CD45RA− markers for HSC purification. With thisimmunophenotype and xenotransplantation assay, we have directly isolatedcord blood HSC activity to fewer cells than in previous reports.

Implications for Human Acute Myeloid Leukemia. Analogous to normalhematopoiesis, human acute myeloid leukemia (AML) is organized as ahierarchy initiated by leukemia stem cells (LSC) that are able toself-renew and give rise to all the cells within the leukemia (Tan etal. (2006) Lab Invest 86, 1203-1207; Wang and Dick (2005) Trends CellBiol 15, 494-501). In a series of xenotransplantation experiments, Dickand colleagues first demonstrated the existence of human AML LSC andlocalized them to the Lin−CD34+CD38− fraction of AML (Bonnet and Dick(1997) Nat Med 3, 730-737; Lapidot et al. (1994) Nature 367, 645-648).Based on these observations, a model was proposed in which HSC are thecell of origin for AML LSC. However, subsequent experiments indicatedthat AML LSC, unlike HSC, do not express CD90 (Miyamoto et al. (2000)Proc Natl Acad Sci USA 97, 7521-7526). There are two hypotheses toaccount for this difference: (1) AML LSC are indeed derived from HSC,but have aberrantly lost expression of CD90, or (2) AML LSC do notderive from HSC but instead come from a downstream progenitor that lacksexpression of CD90.

Evidence supporting the second hypothesis comes from previous studies ofAML1-ETO translocation-associated AML in atom bomb survivors fromHiroshima (Miyamoto et al., 2000). The AML LSC were contained in theLin−CD34+CD38−CD90− fraction (Blair et al. (1997) Blood 89, 3104-3112).However, when the bone marrow of long-term disease-free survivors wasexamined, the AML1-ETO translocation was detected in Lin−CD34+CD38−CD90+non-leukemic HSC. This demonstrates that pre-leukemic genetic changescan take place within HSC, but ultimate transformation to AML LSCrequires additional mutations that do not occur in HSC, but instead takeplace in a CD90− downstream population.

Why does this matter? If the normal counterpart to long-termself-renewing AML LSC is not capable of long-term self-renewal itself,then AML LSC must have undergone mutational or epigenetic activation ofa self-renewal pathway. These changes, when identified, become targetsfor therapeutic intervention to eradicate the LSC. Evidence for aberrantactivation of self-renewal in LSC comes from studies of human blastcrisis CML, where normally non-self-renewing cells transform into LSC inpart through activation of the Wnt/beta-catenin pathway (Jamieson et al.(2004) N Engl J Med 351, 657-667). Through comparisons between AML LSCand the newly identified MPP, genetic and/or epigenetic events than leadto the transformation of MPP into AML LSC are identified.

Experimental Procedures

Human Samples. Normal human bone marrow mononuclear cells were purchasedfrom AllCells Inc. (Emeryville, Calif.). Human cord blood was collectedfrom placentas and/or umbilical cords obtained from the Stanford MedicalCenter, according to an IRB-approved protocol (Stanford IRB #4637).Mononuclear cells were prepared using Ficoll-Paque Plus (GE Healthcare,Fairfield, Conn.), and cryopreserved in 90% FBS/10% DMSO. Allexperiments were conducted with cryopreserved cord blood cells that werethawed and washed with IMDM containing 10% FBS. In some cases, CD34+cells were enriched using MACS (Miltenyi Biotec, Germany) or Robosep(Stem Cell Technologies, Canada) immunomagnetic beads.

Flow Cytometry Analysis and Cell Sorting. A panel of antibodies was usedfor analysis and sorting of progenitor subpopulations, as well aslineage analysis of human chimerism/engraftment, and used to stain cellsuspensions (Supplementary Methods). Cells were either analyzed orsorted using a FACSAria cytometer (BD Biosciences). Analysis of flowcytometry data was performed using FlowJo Software (Treestar, Ashland,Oreg.). Raw engraftment data is provided in Supplementary FIG. 3.Statistical analysis using Student's t-test or Fisher's exact test wasperformed with Microsoft Excel and/or GraphPad Prism (San Diego, Calif.)software.

In Vitro Assays: Cytology, Methylcellulose, and Liquid Culture. Forcytologic analysis, sorted cells were centrifuged onto slides using aShandon Cytocentrifuge 4 (Thermo Scientific, Waltham, Mass.), andstained with Wright-Giemsa. Photomicrographs were taken using a 100×objective under oil.

Methylcellulose colony formation was assayed by clone-sorting singlecells into individual wells of a 96-well plate, each containing 100 μlof complete methylcellulose (Methocult GF+ H4435, Stem CellTechnologies). Plates were incubated for 12-14 days at 37° C., thenscored based on morphology. All colonies were harvested, dissociated byresuspending in sterile PBS, and replated into individual wells of a24-well plate, each containing 500 μl of complete methylcellulose.Plates were incubated for 12-14 days at 37° C., after which replatingwas determined by assessing colony formation. Statistical analysis usingStudent's t-test was performed with Microsoft Excel and/or GraphPadPrism (San Diego, Calif.) software.

In vitro proliferation was assayed by clone-sorting single cells intoindividual wells of a 96-well plate, each containing 100 μl of StemSpanmedia (Stem Cell Technologies), supplemented with 40 μg/ml human LDL(Sigma-Aldrich) and cytokines (R&D Systems, Minneapolis, Minn.): 100ng/ml Flt-3 ligand, 100 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3, and 20ng/ml IL-6. Plates were incubated for 14 days at 37° C., after whichlive cells were counted by trypan blue exclusion. For in vitrodifferentiation assays, cells were sorted in bulk into this same culturemedia and incubated for 3-4 days at 37° C., after which cells wereharvested and analyzed by flow cytometry. Statistical analysis usingStudent's t-test was performed with Microsoft Excel and/or GraphPadPrism (San Diego, Calif.) software.

Mouse Transplantation. NOD.Cg-Prkdc^(scid)II2rg^(tm1wjl)/SzJ mice (NOG)were obtained from The Jackson Laboratory (Bar Harbor, Me.) and bred ina Specific Pathogen-Free environment per Stanford Administrative Panelon Laboratory Animal Care guidelines (Protocol 10725). P0-P2 newbornpups were conditioned with 100 rads of gamma irradiation up to 24 hoursprior to transplantation (Ishikawa et al., 2005). Desired cells wereresuspended in 20-40 ul of PBS containing 2% FBS and transplantedintravenously via the anterior facial vein using a 30 or 31 gaugeneedle. For secondary transplants, human CD34+ bone marrow cells fromprimary engrafted mice were enriched using MACS immunomagnetic beads(Miltenyi Biotec), and transplanted into newborn NOG recipients.

Example 2 Identification of Cell Surface Molecules PreferentiallyExpressed on Human Acute Myeloid Leukemia Stem Cells Compared to TheirNormal Counterparts

Prospective Identification of a Human Multipotent Progenitor, the Cellof Origin for AML LSC. Identification of cell surface molecules that arepreferentially expressed on AML LSC would be greatly facilitated bydetermining the cell within the normal hematopoietic hierarchy thatundergoes transformation to become an AML LSC. The prevailing view inthe field has been that AML LSC arise out of hematopoietic stem cells(HSC), since both stem cell populations are enriched in Lin−CD34+CD38−cells. However, human HSC have been shown to express CD90, while AML LSCare CD90−. Furthermore, HSC from long-term remission t(8;21) AMLpatients were found to contain the AML1-ETO translocation product,suggesting that the HSC were pre-leukemic, and that full transformationto AML LSC occurred in a downstream progenitor.

While it is certainly possible that HSC are in fact the cell of originfor AML LSC, and that these cells lose expression of CD90 as aconsequence of transformation, it is also possible that AML LSCoriginate from downstream Lin−CD34+CD38−CD90− cells. We utilized aNOD/SCID/IL-2R gamma null (NOG) newborn xenotransplantation model toassay the function of subpopulations of Lin−CD34+CD38− cord blood,identified on the basis of CD90 and CD45RA expression.Lin−CD34+CD38−CD90+ cells produced long-term multi-lineage engraftmentand formed successful secondary transplants, and therefore containedHSC. Transplantation of purified Lin−CD34+CD38−CD90−CD45RA− cellsresulted in lower levels of multi-lineage engraftment in primaryrecipients, and a statistically significant reduced ability to formlong-term secondary transplants. In fact, with transplantation of 50purified cells, these cells failed to long-term engraft, unlike theLin−CD34+CD38−CD90+ HSC. Thus, Lin−CD34+CD38−CD90−CD45RA− cells aremultipotent and possess limited self-renewal ability. These cells aretermed multipotent progenitors (MPP) and represent the possible cell oforigin of AML LSC.

Use of Gene Expression Profiling to Identify Cell Surface MoleculesPreferentially Expressed on AML LSC Compared to Their NormalCounterparts, HSC and MPP. Cell surface molecules preferentiallyexpressed on human acute myeloid leukemia stem cells (AML LSC) comparedto their normal counterparts have therapeutic applications outlinedbelow. One strategy to identify such molecules has been to generate geneexpression profiles of AML LSC and normal HSC and MPP, and compare themfor differentially expressed genes.

Normal bone marrow HSC and MPP (n=4) and AML LSC (n=9) were purified byFACS. Total RNA was prepared, amplified, and hybridized to Affymetrixhuman DNA microarrays. Statistical analysis identified 4037 genesdifferentially expressed between HSC and LSC, and 4208 genesdifferentially expressed between MPP and LSC, with p<0.05 and a FalseDiscovery Rate of 5% (FIG. 8A). Investigation of these differentiallyexpressed genes identified 288 and 318 cell surface moleculespreferentially expressed in AML LSC by at least 2-fold compared to HSCand MPP, respectively. Selected members of this list, including manywith the greatest preferential expression in AML LSC are indicated (FIG.8B, Table 1).

TABLE 1 Fold Change Genbank Gene Symbol Description 94.34 M27331 TRGC2 Tcell receptor gamma constant 2 57.47 NM_005816 CD96 CD96 antigen 47.17AI862120 MAMDC2 MAM domain containing 2 32.36 AF348078 SUCNR1 succinatereceptor 1 32.05 M16768 TRGC2 T cell receptor gamma constant 2 30.96NM_002182 IL1RAP interleukin 1 receptor accessory protein 29.85 M13231TRGC2 T cell receptor gamma constant 2 27.55 NM_003332 TYROBP TYROprotein tyrosine kinase binding protein 26.88 NM_004271 LY86 lymphocyteantigen 86 20.96 NM_014879 P2RY14 purinergic receptor P2Y, G-proteincoupled, 14 18.38 BC020749 CD96 CD96 antigen 18.38 NM_005048 PTHR2parathyroid hormone receptor 2 17.73 AI625747 ADRB1 Adrenergic, beta-1-,receptor 17.36 NM_015376 RASGRP3 RAS guanyl releasing protein 3 (calciumand DAG-regulated) 16.84 U62027 C3AR1 complement component 3a receptor 114.49 AW025572 HAVCR2 hepatitis A virus cellular receptor 2 12.48AF285447 HCST hematopoietic cell signal transducer 11.92 AI805323 LGR7leucine-rich repeat-containing G protein- coupled receptor 7 11.67NM_001197 BIK BCL2-interacting killer (apoptosis-inducing) 11.53NM_018092 NETO2 neuropilin (NRP) and tolloid (TLL)-like 2 11.07 N74607AQP3 aquaporin 3 10.88 BF439675 CD69 CD69 antigen (p60, early T-cellactivation antigen) 10.48 NM_001769 CD9 CD9 antigen (p24) 10.32 AF167343IL1RAP interleukin 1 receptor accessory protein  9.52 AA814140 C5orf18chromosome 5 open reading frame 18  8.77 NM_005582 CD180 CD180 antigen 7.46 AF039686 GPR34 G protein-coupled receptor 34  7.30 AI056776 ITGA6Integrin, alpha 6  7.19 AJ277151 TNFRSF4 tumor necrosis factor receptorsuperfamily, member 4  6.99 AI738675 SELPLG Selectin P ligand  6.85AA888858 PDE3B Phosphodiesterase 3B, cGMP-inhibited  6.80 AU149572 ADCY2adenylate cyclase 2 (brain)  6.80 NM_002299 LCT lactase  6.58 NM_005296GPR23 G protein-coupled receptor 23  6.45 NM_004106 FCER1G Fc fragmentof IgE, high affinity receptor  6.29 AI741056 SELPLG selectin P ligand 6.25 AW406569 MGC15619  6.06 M81695 ITGAX integrin, alpha X  5.92NM_003494 DYSF dysferlin  5.85 AI860212 PAG1 phosphoprotein associatedwith glycosphingolipid microdomains 1  5.75 NM_013447 EMR2 egf-likemodule containing, mucin-like, hormone receptor-like 2  5.62 NM_017806LIME1 Lck interacting transmembrane adaptor 1  5.62 AK092824 AMNAmnionless homolog (mouse)  5.59 AF345567 GPR174 G protein-coupledreceptor 174  5.29 BC041928 IL1RAP Interleukin 1 receptor accessoryprotein  5.26 L03419 FCGR1A Fc fragment of IgG, high affinity Ia,receptor (CD64); Fc-gamma receptor I B2  5.24 BG230586 SLC7A6 solutecarrier family 7 (cationic amino acid transporter, y + system), member 6 5.18 AF015524 CCRL2 chemokine (C-C motif) receptor-like 2  5.13AA631143 SLC45A3 solute carrier family 45, member 3  5.10 AJ240085 TRAT1T cell receptor associated transmembrane adaptor 1  5.05 AW183080 GPR92G protein-coupled receptor 92  5.03 NM_002120 HLA-DOB majorhistocompatibility complex, class II, DO beta  5.03 NM_015364 LY96lymphocyte antigen 96  4.90 NM_020399 GOPC golgi associated PDZ andcoiled-coil motif containing  4.88 AK026133 SEMA4B semaphorin  4.88BC041664 VMD2 vitelliform macular dystrophy 2  4.85 NM_152592 C14orf49chromosome 14 open reading frame 49  4.85 AA923524 RASGRP4 RAS guanylreleasing protein 4  4.85 BC008777 ITGAL integrin, alpha L)  4.67AF014403 PPAP2A phosphatidic acid phosphatase type 2A  4.65 AK097698SORCS2 Sortilin-related VPS10 domain containing receptor 2  4.63 X14355FCGR1A Fc fragment of IgG, high affinity Ia, receptor (CD64)  4.55NM_001629 ALOX5AP arachidonate 5-lipoxygenase-activating protein  4.50AU155968 C18orf1 chromosome 18 open reading frame 1  4.44 AK075092HERV-FRD HERV-FRD provirus ancestral Env polyprotein  4.42 NM_020960GPR107 G protein-coupled receptor 107  4.37 BC000039 FAM26B family withsequence similarity 26, member B  4.35 NM_153701 IL12RB1 interleukin 12receptor, beta 1  4.35 AI762344 PTGER1 prostaglandin E receptor 1(subtype EP1), 42 kDa  4.31 NM_006459 SPFH1 SPFH domain family, member 1 4.27 NM_003126 SPTA1 spectrin, alpha, erythrocytic 1 (elliptocytosis 2) 4.22 AL518391 AQP1 aquaporin 1 (channel-forming integral protein, 28kDa)  4.12 AK026188 PCDHGC3 protocadherin gamma subfamily C  4.10AU146685 EDG2 Endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2  4.05 BE673587 SLC14A1 Solute carrier family14 (urea transporter), member 1 (Kidd blood group)  4.02 BF129969 TSPAN2tetraspanin 2  4.00 AW243272 KCNK5 Potassium channel, subfamily K,member 5  3.98 T68858 DHRS3 Dehydrogenase/reductase (SDR family) member3  3.94 AI827849 VTI1A Vesicle transport through interaction witht-SNAREs homolog 1A (yeast)  3.86 AL134012 NRXN2 Neurexin 2  3.83BG230614 CD47 CD47 antigen  3.80 AI869717 MGC15523 MGC15523  3.80AI458583 SIMP Source of immunodominant MHC-associated peptides  3.79NM_002183 IL3RA interleukin 3 receptor, alpha (low affinity)  3.79AA608820 NRXN2 neurexin 2  3.73 NM_000206 IL2RG interleukin 2 receptor 3.72 BC002737 VAMP2 synaptobrevin 2  3.72 BC005884 BID BH3 interactingdomain death agonist; BH3 interacting domain death agonist  3.68AI688418 PLXNA2 plexin A2  3.68 BC003105 PTP4A3 protein tyrosinephosphatase type IVA, member 3  3.68 NM_001772 CD33 CD33 antigen (gp67) 3.65 BC007524 SPAG9 sperm associated antigen 9  3.64 AI344200 SLC25A35solute carrier family 25, member 35  3.64 B0005253 KLHL20 kelch-like 20(Drosophila)  3.60 AI335263 NETO2 neuropilin (NRP) and tolloid(TLL)-like 2  3.58 BF381837 C20orf52 chromosome 20 open reading frame 52 3.51 NM_002886 RAP2A RAP2A  3.50 NM_007063 TBC1D8 TBC1 domain family,member 8 (with GRAM domain)  3.45 AK027160 BCL2L11 BCL2-like 11(apoptosis facilitator)  3.44 BF055366 EDG2 endothelial differentiation,lysophosphatidic acid G- protein-coupled receptor, 2  3.42 NM_003608GPR65 G protein-coupled receptor 65  3.41 AI675453 PLXNA3 plexin A3 3.40 AV734194 DPP8 dipeptidylpeptidase 8  3.38 B0000232 C5orf18chromosome 5 open reading frame 18  3.36 B0001956 KIAA1961 KIAA1961 gene 3.34 NM_013332 HIG2 hypoxia-inducible protein 2  3.31 BC029450 SLC33A1Solute carrier family 33 (acetyl-CoA transporter), member 1  3.30AW008505 C18orf1 chromosome 18 open reading frame 1  3.29 BF693956 CD47CD47 antigen  3.28 BF677986 KIAA1961 KIAA1961 gene  3.27 AI433691CACNA2D4 calcium channel, voltage-dependent, alpha 2/delta subunit 4 3.26 AB014573 NPHP4 nephronophthisis 4  3.25 AL582804 LY9 lymphocyteantigen 9  3.25 BG236280 CD86 CD86 antigen  3.24 AA639289 5LC26A7 Solutecarrier family 26, member 7  3.24 NM_005211 CSF1R colony stimulatingfactor 1 receptor  3.24 AI051254 TRPM2 transient receptor potentialcation channel, subfamily M, member 2  3.23 AW292816 ABHD2 abhydrolasedomain containing 2  3.23 BC040275 RASGRF1 Ras protein-specific guaninenucleotide-releasing factor 1  3.22 NM_021911 GABRB2 gamma-aminobutyricacid (GABA) A receptor, beta 2  3.19 AI660619 SLC7A6 solute carrierfamily 7 (cationic amino acid transporter, y + system), member 6  3.19NM_001860 SLC31A2 solute carrier family 31 (copper transporters), member2  3.18 NM_015680 C2orf24 chromosome 2 open reading frame 24  3.17AW058600 SLC36A1 solute carrier family 36  3.16 AU145049 HIP1 Huntingtininteracting protein 1  3.15 NM_005770 SERF2 small EDRK-rich factor 2 3.15 NM_003566 EEA1 Early endosome antigen 1, 162 kD  3.14 NM_020041SLC2A9 solute carrier family 2 (facilitated glucose transporter), member9  3.14 W90718 SLC24A4 solute carrier family 24  3.13 AI423165 TICAM2toll-like receptor adaptor molecule 2  3.12 AI674647 SPPL2A signalpeptide peptidase-like 2A  3.11 NM_004121 GGTLA1gamma-glutamyltransferase-like activity 1  3.10 NM_004546 NDUFB2 NADHdehydrogenase (ubiquinone) 1 beta subcomplex, 2, 8 kDa  3.05 X15786 RETret proto-oncogene (multiple endocrine neoplasia and medullary thyroidcarcinoma 1, Hirschsprung disease)  3.05 AF181660 MPZL1 myelin proteinzero-like 1  3.05 BG230614 CD47 CD47 antigen (Rh-related antigen,integrin-associated signal transducer)  3.00 AI571996 STAM2 signaltransducing adaptor molecule (SH3 domain and ITAM motif) 2  2.99NM_000201 ICAM1 intercellular adhesion molecule 1 (CD54), humanrhinovirus receptor  2.93 NM_025244 TSGA10 testis specific, 10  2.93AU147538 PRKCE Protein kinase C, epsilon  2.92 NM_024576 OGFRL1 opioidgrowth factor receptor-like 1  2.91 AI248055 ABCC4 ATP-binding cassette,sub-family C (CFTR/MRP), member 4  2.86 AA503877 CEPT1Choline/ethanolamine phosphotransferase 1  2.84 BC030993 FLJ21127Hypothetical protein FLJ21127  2.82 AA829818 LY86 Lymphocyte antigen 86 2.82 NM_001859 SLC31A1 solute carrier family 31 (copper transporters),member 1  2.81 M74721 CD79A CD79A antigen (immunoglobulin-associatedalpha)  2.79 AI986112 MGAT4B Mannosyl (alpha-1,3-)-glycoproteinbeta-1,4-N- acetylglucosaminyltransferase, isoenzyme B  2.79 NM_030930UNC93B1 unc-93 homolog B1 (C. elegans); unc-93 homolog B1 (C. elegans) 2.79 X74039 PLAUR plasminogen activator, urokinase receptor  2.78BF514291 LY86 Lymphocyte antigen 86  2.75 BC005253 KLHL20 kelch-like 20(Drosophila)  2.73 AB036432 AGER advanced glycosylation endproduct-specific receptor  2.71 NM_007245 ATXN2L ataxin 2-like  2.71NM_016072 GOLT1B golgi transport 1 homolog B (S. cerevisiae)  2.71AI453548 ZDHHC8 zinc finger, DHHC-type containing 8  2.70 AI636233 TMEM8transmembrane protein 8 (five membrane- spanning domains)  2.69 BE502509T3JAM TRAF3 interacting protein 3  2.69 AW117765 PEX13 peroxisomebiogenesis factor 13  2.69 AW052216 IL17RB Interleukin 17 receptor B 2.67 NM_003853 IL18RAP interleukin 18 receptor accessory protein  2.66NM_002490 NDUFA6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex,6,14 kDa  2.65 NM_016639 TNFRSF12A tumor necrosis factor receptorsuperfamily, member 12A  2.65 AI363185 FLJ20255 Hypothetical proteinFLJ20255  2.65 NM_052931 SLAMF6 SLAM family member 6  2.65 AW571669TNFRSF19L tumor necrosis factor receptor superfamily, member 19-like 2.64 AA654142 CEECAM1 cerebral endothelial cell adhesion molecule 1 2.62 AW510783 TMEM63A transmembrane protein 63A  2.61 W95007 ACSL4Acyl-CoA synthetase long-chain family member 4  2.60 S76475 NTRK3neurotrophic tyrosine kinase, receptor, type 3  2.60 AJ130713 SIGLEC7sialic acid binding Ig-like lectin 7  2.56 NM_003775 EDG6 endothelialdifferentiation, G-protein-coupled receptor 6  2.55 AI978986 MAMDC4 MAMdomain containing 4  2.54 AF010447 MR1 major histocompatibility complex,class I-related  2.54 NM_006068 TLR6 toll-like receptor 6  2.53 AF041811NTRK3 neurotrophic tyrosine kinase, receptor, type 3  2.53 AW953521SERF2; small EDRK-rich factor 2; Huntingtin interacting HYPK protein K 2.51 AW293276 CD53 CD53 antigen  2.49 AK023058 PLXNA2 Plexin A2  2.49AI125204 C6orf128 chromosome 6 open reading frame 128  2.49 NM_000392ABCC2 ATP-binding cassette, sub-family C (CFTR/MRP), member 2  2.46B0032474 TIRAP toll-interleukin 1 receptor (TIR) domain containingadaptor protein  2.44 NM_031211 IMAA SLC7A5 pseudogene  2.44 AI797836CD5 CD5 antigen (p56-62)  2.41 W72082 C1QR1 complement component 1  2.40AA708616 DPP9 dipeptidylpeptidase 9  2.40 BM987094 DLGAP4 discs, large(Drosophila) homolog-associated protein 4  2.40 AL713719 LOC283501ATPase, Class VI, type 11A  2.39 AI628734 PRLR prolactin receptor  2.39NM_012110 CHIC2 cysteine-rich hydrophobic domain 2  2.38 AK022002 TFR2transferrin receptor 2  2.37 NM_001555 IGSF1 immunoglobulin superfamily,member 1  2.36 AA426091 C19orf15 chromosome 19 open reading frame 15 2.36 BE547542 GOPC golgi associated PDZ and coiled-coil motifcontaining  2.36 NM_004231 ATP6V1F ATPase, H + transporting, lysosomal14 kDa, V1 subunit F  2.36 AJ130712 SIGLEC7 sialic acid binding Ig-likelectin 7  2.36 NM_017905 TMCO3 transmembrane and coiled-coil domains 3 2.35 AB054985 CACNB1 calcium channel, voltage-dependent, beta 1 subunit 2.35 NM_005003 NDUFAB1 NADH dehydrogenase (ubiquinone) 1, alpha/betasubcomplex, 1, 8 kDa  2.35 NM_001251 CD68 CD68 antigen  2.35 AA700869PSCD2 Pleckstrin homology, Sec7 and coiled-coil domains 2 (cytohesin-2) 2.35 U94903 CD44 CD44 antigen (homing function and Indian blood groupsystem)  2.35 NM_003841 TNFRSF10C tumor necrosis factor receptorsuperfamily, member 10c, decoy without an intracellular domain  2.33NM_004541 NDUFA1 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1,7.5 kDa  2.33 BE567130 KLRK1 Killer cell lectin-like receptor subfamilyK, member 1  2.31 NM_017460 CYP3A4 cytochrome P450, family 3, subfamilyA, polypeptide 4  2.31 AI339536 DSC1 Desmocollin 1  2.31 NM_001783 CD79ACD79A antigen (immunoglobulin-associated alpha); CD79A antigen(immunoglobulin-associated alpha)  2.30 AA333161 VTI1A vesicle transportthrough interaction with t-SNAREs homolog 1A (yeast)  2.30 AW134823 CD6CD6 antigen ; CD6 antigen  2.30 AL137537 ATP8B2 ATPase, Class I, type8B, member 2  2.29 AI671983 SLC2A9 solute carrier family 2 (facilitatedglucose transporter), member 9  2.29 AA018187 C22orf3 chromosome 22 openreading frame 3  2.29 AL117415 ADAM33 ADAM metallopeptidase domain 33 2.29 NM_002588 PCDHGC3 protocadherin gamma subfamily C  2.29 NM_020960GPR107 G protein-coupled receptor 107  2.29 AK074635 GENX-3414Genethonin 1  2.29 BE138575 ITGB5 Integrin, beta 5  2.28 NM_003830SIGLEC5 sialic acid binding Ig-like lectin 5; sialic acid bindingIg-like lectin 5  2.28 NM_013319 UBIAD1 UbiA prenyltransferase domaincontaining 1  2.28 M63889 FGFR1 fibroblast growth factor receptor 1(fms-related tyrosine kinase 2, Pfeiffer syndrome)  2.27 H67156 MSCPSolute carrier family 25, member 37  2.27 BC006215 SMEK2 KIAA1387protein; KIAA1387 protein  2.27 AL109653 SLITRK2 SLIT and NTRK-likefamily, member 2  2.27 NM_007011 ABHD2 abhydrolase domain containing 2 2.26 AI767210 MGC11332 Hypothetical protein MGC11332  2.26 BF723605NRCAM Neuronal cell adhesion molecule  2.26 R08129 CDA08 T-cellimmunomodulatory protein  2.26 AF052059 SEL1L sel-1 suppressor oflin-12-like (C. elegans)  2.26 NM_005729 PPIF peptidylprolyl isomerase F(cyclophilin F)  2.25 BE858032 ARL2L1 ADP-ribosylation factor-like2-like 1  2.25 AI950390 C14orf118 Chromosome 14 open reading frame 118 2.24 NM_017767 SLC39A4 solute carrier family 39 (zinc transporter),member 4  2.24 AL110273 SPTAN1 Spectrin, alpha, non-erythrocytic 1(alpha-fodrin)  2.24 AI077660 CDA08 T-cell immunomodulatory protein 2.23 AA488687 SLC7A11 solute carrier family 7, (cationic amino acidtransporter, y + system) member 11  2.23 NM_000634 IL8RA interleukin 8receptor, alpha  2.22 AL390177 MGC34032 Solute carrier family 44, member5  2.21 NM_001531 MR1 major histocompatibility complex, class I-related 2.21 NM_003183 ADAM17 ADAM metallopeptidase domain 17 (tumor necrosisfactor, alpha, converting enzyme)  2.20 AC003999 SCAP2 src familyassociated phosphoprotein 2  2.20 BC014416 SLC33A1 solute carrier family33 (acetyl-CoA transporter), member 1  2.20 AF226731 ADORA3 adenosine A3receptor  2.19 AI608725 ICAM1 intercellular adhesion molecule 1 (CD54),human rhinovirus receptor  2.19 U41163 SLC6A8; solute carrier family 6(neurotransmitter transporter, FLJ43855 creatine), member 8; similar tosodium- and chloride-dependent creatine transporter  2.19 AU147799LRRC15 leucine rich repeat containing 15  2.18 AW337166 LOC255104Transmembrane and coiled-coil domains 4  2.18 NM_006505 PVR poliovirusreceptor  2.18 AI638420 CLIC4 chloride intracellular channel 4  2.18AI167482 SCUBE3 Signal peptide, CUB domain, EGF-like 3  2.18 AI739514HAS3 hyaluronan synthase 3  2.18 NM_005971 FXYD3 FXYD domain containingion transport regulator 3  2.17 AL022398 TRAF3IP3 TRAF3 interactingprotein 3  2.17 U90940 FCGR2C Fc fragment of IgG, low affinity IIc,receptor for (CD32)  2.16 BCO23540 SORCS1 Sortilin-related VPS10 domaincontaining receptor 1  2.16 AV713913 OSTM1 osteopetrosis associatedtransmembrane protein 1  2.15 NM_024505 NOX5 NADPH oxidase, EF-handcalcium binding domain 5  2.15 BC006178 SEC22L3 SEC22 vesicletrafficking protein-like 3 (S. cerevisiae); SEC22 vesicle traffickingprotein-like 3 (S. cerevisiae)  2.15 BG151527 GRIK5 glutamate receptor,ionotropic, kainate 5  2.14 AW001754 NEGRI neuronal growth regulator 1 2.14 NM_013979 BNIP1 BCL2/adenovirus E1B 19 kDa interacting protein 1 2.14 NM_018643 TREM1 triggering receptor expressed on myeloid cells 1 2.12 NM_005284 GPR6 G protein-coupled receptor 6  2.11 AA454190 ZDHHC20zinc finger, DHHC-type containing 20  2.11 AB048796 TMPRSS13transmembrane protease, serine 13  2.11 AL044520 NYD-SP21 testesdevelopment-related NYD-SP21  2.11 BE463930 TMAP1 Matrix-remodellingassociated 7  2.10 NM_152264 SLC39A13 solute carrier family 39 (zinctransporter), member 13  2.08 AL530874 EPHB2 EPH receptor B2  2.07NM_018668 VPS33B vacuolar protein sorting 33B (yeast)  2.07 NM_024531GPR172A G protein-coupled receptor 172A  2.07 NM_023038 ADAM19 ADAMmetallopeptidase domain 19 (meltrin beta)  2.07 BC001281 TNFRSF10B tumornecrosis factor receptor superfamily, member 10b  2.07 AF217749 PCDHB9protocadherin beta 9  2.06 AB030077 FGFR2 fibroblast growth factorreceptor 2 (bacteria-expressed kinase, keratinocyte growth factorreceptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffersyndrome, Jackson-Weiss syndrome)  2.06 AL137432 SUSD1 sushi domaincontaining 1  2.05 NM_004518 KCNQ2 potassium voltage-gated channel,KQT-like subfamily, member 2  2.04 AI672363 VPS33B vacuolar proteinsorting 33B (yeast)  2.04 NM_006671 SLC1A7 solute carrier family 1(glutamate transporter), member 7  2.03 AA215519 DLGAP1 Discs, large(Drosophila) homolog-associated protein 1  2.02 NM_004648 PTPNS1 proteintyrosine phosphatase, non-receptor type substrate 1  2.02 NM_002564P2RY2 purinergic receptor P2Y, G-protein coupled, 2  2.01 BF511678SCUBE3 Signal peptide, CUB domain, EGF-like 3  2.01 BC013385 CLEC7AC-type lectin domain family 7, member A

TABLE 2 Fold Change Genbank Gene Symbol Description 57.47 NM_005816 CD96CD96 antigen 32.36 AF348078 SUCNR1 succinate receptor 1 30.96 NM_002182IL1RAP interleukin 1 receptor accessory protein 27.55 NM_003332 TYROBPTYRO protein tyrosine kinase binding protein 26.88 NM_004271 LY86lymphocyte antigen 86 20.96 NM_014879 P2RY14 purinergic receptor P2Y,G-protein coupled, 14 18.38 NM_005048 PTHR2 parathyroid hormone receptor2 17.73 AI625747 ADRB1 Adrenergic, beta-1-, receptor 17.36 NM_015376RASGRP3 RAS guanyl releasing protein 3 (calcium and DAG-regulated) 16.84U62027 C3AR1 complement component 3a receptor 1 14.49 AW025572 HAVCR2hepatitis A virus cellular receptor 2 12.48 AF285447 HCST hematopoieticcell signal transducer 11.92 AI805323 LGR7 leucine-richrepeat-containing G protein-coupled receptor 7 11.67 NM_001197 BIKBCL2-interacting killer (apoptosis-inducing) 11.53 NM_018092 NETO2neuropilin (NRP) and tolloid (TLL)-like 2 11.07 N74607 AQP3 aquaporin 310.48 NM_001769 CD9 CD9 antigen (p24)  8.77 NM_005582 CD180 CD180antigen  7.46 AF039686 GPR34 G protein-coupled receptor 34  7.19AJ277151 TNFRSF4 tumor necrosis factor receptor superfamily, member 4 6.85 AA888858 PDE3B Phosphodiesterase 3B, cGMP-inhibited  6.80 AU149572ADCY2 adenylate cyclase 2 (brain)  6.80 NM_002299 LCT lactase  6.58NM_005296 GPR23 G protein-coupled receptor 23  6.45 NM_004106 FCER1G Fcfragment of IgE, high affinity I, receptor for; gamma polypeptide  6.25AW406569 MGC15619 hypothetical protein MGC15619  6.06 M81695 ITGAXintegrin, alpha X (antigen CD11C (p150), alpha polypeptide)  5.92NM_003494 DYSF dysferlin, limb girdle muscular dystrophy 2B (autosomalrecessive)  5.75 NM_013447 EMR2 egf-like module containing, mucin-like,hormone receptor-like 2  5.62 NM_017806 LIME1 Lck interactingtransmembrane adaptor 1  5.62 AK092824 AMN Amnionless homolog (mouse) 5.59 AF345567 GPR174 G protein-coupled receptor 174  5.26 L03419FCGR1A; Fc fragment of IgG, high affinity Ia, receptor (CD64); LOC440607Fc-gamma receptor I B2  5.18 AF015524 CCRL2 chemokine (C-C motif)receptor-like 2  5.13 AA631143 SLC45A3 solute carrier family 45, member3  5.10 AJ240085 TRAT1 T cell receptor associated transmembrane adaptor1  5.05 AW183080 GPR92 G protein-coupled receptor 92  5.03 NM_002120HLA-DOB major histocompatibility complex, class II, DO beta  5.03NM_015364 LY96 lymphocyte antigen 96

TABLE 3 Fold Change Genbank Gene Symbol Description 57.47 NM_005816 CD96CD96 antigen 32.36 AF348078 SUCNR1 succinate receptor 1 30.96 NM_002182IL1RAP interleukin 1 receptor accessory protein 27.55 NM_003332 TYROBPTYRO protein tyrosine kinase binding protein 26.88 NM_004271 LY86lymphocyte antigen 86 20.96 NM_014879 P2RY14 purinergic receptor P2Y,G-protein coupled, 14 18.38 NM_005048 PTHR2 parathyroid hormone receptor2 17.73 AI625747 ADRB1 Adrenergic, beta-1-, receptor 17.36 NM_015376RASGRP3 RAS guanyl releasing protein 3 (calcium and DAG-regulated) 16.84U62027 C3AR1 complement component 3a receptor 1 14.49 AW025572 HAVCR2hepatitis A virus cellular receptor 2 12.48 AF285447 HCST hematopoieticcell signal transducer 11.92 AI805323 LGR7 leucine-richrepeat-containing G protein-coupled receptor 7 11.67 NM_001197 BIKBCL2-interacting killer (apoptosis-inducing) 11.53 NM_018092 NETO2neuropilin (NRP) and tolloid (TLL)-like 2 11.07 N74607 AQP3 aquaporin 310.88 BF439675 CD69 CD69 antigen (p60, early T-cell activation antigen)10.48 NM_001769 CD9 CD9 antigen (p24)  9.52 AA814140 C5orf18 chromosome5 open reading frame 18  8.77 NM_005582 CD180 CD180 antigen  7.46AF039686 GPR34 G protein-coupled receptor 34  7.30 AI056776 ITGA6Integrin, alpha 6  7.19 AJ277151 TNFRSF4 tumor necrosis factor receptorsuperfamily, member 4  6.99 AI738675 SELPLG Selectin P ligand  6.85AA888858 PDE3B Phosphodiesterase 3B, cGMP-inhibited  6.80 AU149572 ADCY2adenylate cyclase 2 (brain)  6.80 NM_002299 LCT lactase  6.58 NM_005296GPR23 G protein-coupled receptor 23  6.45 NM_004106 FCER1G Fc fragmentof IgE, high affinity I, receptor for; gamma polypeptide  6.25 AW406569MGC15619 hypothetical protein MGC15619  6.06 M81695 ITGAX integrin,alpha X (antigen CD110 (p150), alpha polypeptide)  5.92 NM_003494 DYSFdysferlin, limb girdle muscular dystrophy 2B (autosomal recessive)  5.85AI860212 PAG1 phosphoprotein associated with glycosphingolipidmicrodomains 1  5.75 NM_013447 EMR2 egf-like module containing,mucin-like, hormone receptor-like 2  5.62 NM_017806 LIME1 Lckinteracting transmembrane adaptor 1  5.62 AK092824 AMN Amnionlesshomolog (mouse)  5.59 AF345567 GPR174 G protein-coupled receptor 174 5.26 L03419 FCGR1A; Fc fragment of IgG, high affinity Ia, receptorLOC440607 (CD64); Fc-gamma receptor I B2  5.24 BG230586 SLC7A6 solutecarrier family 7 (cationic amino acid transporter, y + system), member 6 5.18 AF015524 CCRL2 chemokine (C-C motif) receptor-like 2  5.13AA631143 SLC45A3 solute carrier family 45, member 3  5.10 AJ240085 TRAT1T cell receptor associated transmembrane adaptor 1  5.05 AW183080 GPR92G protein-coupled receptor 92  5.03 NM_002120 HLA-DOB majorhistocompatibility complex, class II, DO beta  5.03 NM_015364 LY96lymphocyte antigen 96  4.90 NM_020399 GOPC golgi associated PDZ andcoiled-coil motif containing  4.88 AK026133 SEMA4B sema domain,immunoglobulin domain (Ig), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 4B  4.88 BC041664 VMD2 vitelliformmacular dystrophy 2 (Best disease, bestrophin)  4.85 NM_152592 C14orf49chromosome 14 open reading frame 49  4.85 AA923524 RASGRP4 RAS guanylreleasing protein 4  4.85 B0008777 ITGAL integrin, alpha L (antigenCD11A (p180)  4.67 AF014403 PPAP2A phosphatidic acid phosphatase type 2A 4.65 AK097698 SORCS2 Sortilin-related VPS10 domain containing receptor2  4.63 X14355 FCGR1A Fc fragment of IgG, high affinity Ia, receptor(CD64)  4.55 NM_001629 ALOX5AP arachidonate 5-lipoxygenase-activatingprotein  4.50 AU155968 C18orf1 chromosome 18 open reading frame 1  4.44AK075092 HERV-FRD HERV-FRD provirus ancestral Env polyprotein  4.42NM_020960 GPR107 G protein-coupled receptor 107  4.37 BC000039 FAM26Bfamily with sequence similarity 26, member B  4.35 NM_153701 IL12RB1interleukin 12 receptor, beta 1  4.35 AI762344 PTGER1 prostaglandin Ereceptor 1 (subtype EP1), 42 kDa  4.31 NM_006459 SPFH1 SPFH domainfamily, member 1  4.27 NM_003126 SPTA1 spectrin, alpha, erythrocytic 1(elliptocytosis 2)  4.22 AL518391 AQP1 aquaporin 1 (channel-formingintegral protein, 28 kDa)  4.12 AK026188 PCDHGC3 protocadherin gammasubfamily C  4.10 AU146685 EDG2 Endothelial differentiation,lysophosphatidic acid G-protein-coupled receptor, 2  4.05 BE673587SLC14A1 Solute carrier family 14 (urea transporter), member 1 (Kiddblood group)  4.02 BF129969 TSPAN2 tetraspanin 2  4.00 AW243272 KCNK5Potassium channel, subfamily K, member 5  3.98 T68858 DHRS3Dehydrogenase/reductase (SDR family) member 3  3.94 AI827849 VTI1AVesicle transport through interaction with t-SNAREs homolog 1A (yeast) 3.86 AL134012 NRXN2 Neurexin 2  3.83 BG230614 CD47 CD47 antigen(Rh-related antigen, integrin- associated signal transducer)  3.80AI869717 MGC15523 hypothetical protein MGC15523  3.80 AI458583 SIMPSource of immunodominant MHC- associated peptides  3.79 NM_002183 IL3RAinterleukin 3 receptor, alpha (low affinity)  3.79 AA608820 NRXN2neurexin 2  3.73 NM_000206 IL2RG interleukin 2 receptor, gamma (severecombined immunodeficiency)  3.72 BC002737 VAMP2 vesicle-associatedmembrane protein 2 (synaptobrevin 2)  3.72 BC005884 BID BH3 interactingdomain death agonist; BH3 interacting domain death agonist  3.68AI688418 PLXNA2 plexin A2  3.68 BC003105 PTP4A3 protein tyrosinephosphatase type IVA, member 3  3.68 NM_001772 CD33 CD33 antigen (gp67) 3.66 AI955119 VAMP2 vesicle-associated membrane protein 2(synaptobrevin 2)  3.65 B0007524 SPAG9 sperm associated antigen 9  3.64AI344200 SLC25A35 solute carrier family 25, member 35  3.64 BC005253KLHL20 kelch-like 20 (Drosophila)  3.58 BF381837 C20orf52 chromosome 20open reading frame 52  3.51 NM_002886 RAP2A; RAP2A, member of RASoncogene family; RAP2B RAP2B, member of RAS oncogene family  3.50NM_007063 TBC1D8 TBC1 domain family, member 8 (with GRAM domain)  3.45AK027160 BCL2L11 BCL2-like 11 (apoptosis facilitator)  3.44 BF055366EDG2 endothelial differentiation, lysophosphatidic acidG-protein-coupled receptor, 2  3.42 NM_003608 GPR65 G protein-coupledreceptor 65  3.41 AI675453 PLXNA3 plexin A3  3.40 AV734194 DPP8dipeptidylpeptidase 8  3.36 BC001956 KIAA1961 KIAA1961 gene  3.34NM_013332 HIG2 hypoxia-inducible protein 2  3.31 BC029450 SLC33A1 Solutecarrier family 33 (acetyl-CoA transporter), member 1  3.28 BF677986KIAA1961 KIAA1961 gene  3.27 AI433691 CACNA2D4 calcium channel,voltage-dependent, alpha 2/ delta subunit 4  3.26 AB014573 NPHP4nephronophthisis 4  3.25 AL582804 LY9 lymphocyte antigen 9  3.25BG236280 CD86 CD86 antigen (CD28 antigen ligand 2, B7-2 antigen)  3.24AA639289 SLC26A7 Solute carrier family 26, member 7  3.24 NM_005211CSF1R colony stimulating factor 1 receptor, formerly McDonough felinesarcoma viral (v-fms) oncogene homolog; colony stimulating factor 1receptor, formerly McDonough feline sarcoma viral (v-fms) oncogenehomolog  3.24 AI051254 TRPM2 transient receptor potential cationchannel, subfamily M, member 2  3.23 AW292816 ABHD2 abhydrolase domaincontaining 2  3.23 B0040275 RASGRF1 Ras protein-specific guaninenucleotide- releasing factor 1  3.22 NM_021911 GABRB2 gamma-aminobutyricacid (GABA) A receptor, beta 2  3.19 AI660619 SLC7A6 solute carrierfamily 7 (cationic amino acid transporter, y + system), member 6  3.19NM_001860 SLC31A2 solute carrier family 31 (copper transporters), member2  3.18 NM_015680 C2orf24 chromosome 2 open reading frame 24  3.17AW058600 SLC36A1 solute carrier family 36 (proton/amino acid symporter),member 1  3.16 AU145049 HIP1 Huntingtin interacting protein 1  3.15NM_005770 SERF2 small EDRK-rich factor 2  3.15 NM_003566 EEA1 Earlyendosome antigen 1, 162 kD  3.14 NM_020041 SLC2A9 solute carrier family2 (facilitated glucose transporter), member 9  3.14 W90718 SLC24A4solute carrier family 24 (sodium/potassium/calcium exchanger), member 4 3.13 AI423165 TICAM2 toll-like receptor adaptor molecule 2  3.12AI674647 SPPL2A signal peptide peptidase-like 2A  3.11 NM_004121 GGTLA1gamma-glutamyltransferase-like activity 1  3.10 NM_004546 NDUFB2 NADHdehydrogenase (ubiquinone) 1 beta subcomplex, 2, 8 kDa  3.05 X15786 RETret proto-oncogene (multiple endocrine neoplasia and medullary thyroidcarcinoma 1, Hirschsprung disease)  3.05 AF181660 MPZL1 myelin proteinzero-like 1  3.00 AI571996 STAM2 signal transducing adaptor molecule(SH3 domain and ITAM motif) 2  2.99 NM_000201 ICAM1 intercellularadhesion molecule 1 (CD54), human rhinovirus receptor  2.93 NM_025244TSGA10 testis specific, 10  2.93 AU147538 PRKCE Protein kinase C,epsilon  2.92 NM_024576 OGFRL1 opioid growth factor receptor-like 1 2.91 AI248055 ABCC4 ATP-binding cassette, sub-family C (CFTR/MRP),member 4  2.86 AA503877 CEPT1 Choline/ethanolamine phosphotransferase 1 2.84 BC030993 FLJ21127 Hypothetical protein FLJ21127  2.82 NM_001859SLC31A1 solute carrier family 31 (copper transporters), member 1  2.81M74721 CD79A CD79A antigen (immunoglobulin-associated alpha)  2.79AI986112 MGAT4B Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isoenzyme B  2.79 NM_030930 UNC93B1unc-93 homolog B1 (C. elegans); unc-93 homolog B1 (C. elegans)  2.79X74039 PLAUR plasminogen activator, urokinase receptor  2.75 BC005253KLHL20 kelch-like 20 (Drosophila)  2.73 AB036432 AGER advancedglycosylation end product-specific receptor  2.71 NM_007245 ATXN2Lataxin 2-like  2.71 NM_016072 GOLT1B golgi transport 1 homolog B (S.cerevisiae)  2.71 AI453548 ZDHHC8 zinc finger, DHHC-type containing 8 2.70 AI636233 TMEM8 transmembrane protein 8 (five membrane- spanningdomains)  2.69 BE502509 T3JAM TRAF3 interacting protein 3  2.69 AW117765PEX13 peroxisome biogenesis factor 13  2.69 AW052216 IL17RB Interleukin17 receptor B  2.67 NM_003853 IL18RAP interleukin 18 receptor accessoryprotein  2.66 NM_002490 NDUFA6 NADH dehydrogenase (ubiquinone) 1 alphasubcomplex, 6, 14 kDa  2.65 NM_016639 TNFRSF12A tumor necrosis factorreceptor superfamily, member 12A  2.65 AI363185 FLJ20255 Hypotheticalprotein FLJ20255  2.65 NM_052931 SLAMF6 SLAM family member 6  2.65AW571669 TNFRSF19L tumor necrosis factor receptor superfamily, member19-like  2.64 AA654142 CEECAM1 cerebral endothelial cell adhesionmolecule 1  2.62 AW510783 TMEM63A transmembrane protein 63A  2.61 W95007ACSL4 Acyl-CoA synthetase long-chain family member 4  2.60 S76475 NTRK3neurotrophic tyrosine kinase, receptor, type 3  2.60 AJ130713 SIGLEC7sialic acid binding Ig-like lectin 7  2.56 NM_003775 EDG6 endothelialdifferentiation, G-protein-coupled receptor 6  2.55 AI978986 MAMDC4 MAMdomain containing 4  2.54 AF010447 MR1 major histocompatibility complex,class I-related  2.54 NM_006068 TLR6 toll-like receptor 6  2.53 AF041811NTRK3 neurotrophic tyrosine kinase, receptor, type 3  2.53 AW953521SERF2; small EDRK-rich factor 2; Huntingtin interacting HYPK protein K 2.51 AW293276 CD53 CD53 antigen  2.49 AK023058 PLXNA2 Plexin A2  2.49AI125204 C6orf128 chromosome 6 open reading frame 128  2.49 NM_000392ABCC2 ATP-binding cassette, sub-family C (CFTR/MRP), member 2  2.46BC032474 TIRAP toll-interleukin 1 receptor (TIR) domain containingadaptor protein  2.44 NM_031211 IMAA; SLC7A5 pseudogene; SLC7A5pseudogene; LOC388221; NPIP-like locus; NPIP-like locus; hypotheticalLOC440345; protein LOC440345; hypothetical protein LOC440354; LOC440345;PI-3-kinase-related kinase SMG-1 LOC595101; pseudogene;PI-3-kinase-related kinase SMG-1 LOC641298 pseudogene;PI-3-kinase-related kinase SMG-1 pseudogene; PI-3-kinase-related kinaseSMG-1 pseudogene; PI-3-kinase-related kinase SMG-1- like locus;PI-3-kinase-related kinase SMG-1- like locus  2.44 AI797836 CD5 CD5antigen (p56-62)  2.41 W72082 C1QR1 complement component 1, qsubcomponent, receptor 1; complement component 1, q subcomponent,receptor 1  2.40 AA708616 DPP9 dipeptidylpeptidase 9  2.40 BM987094DLGAP4 discs, large (Drosophila) homolog-associated protein 4  2.40AL713719 LOC283501 ATPase, Class VI, type 11A  2.39 AI628734 PRLRprolactin receptor  2.39 NM_012110 CHIC2 cysteine-rich hydrophobicdomain 2  2.38 AK022002 TFR2 transferrin receptor 2  2.37 NM_001555IGSF1 immunoglobulin superfamily, member 1  2.36 AA426091 C19orf15chromosome 19 open reading frame 15  2.36 BE547542 GOPC golgi associatedPDZ and coiled-coil motif containing  2.36 NM_004231 ATP6V1F ATPase, H +transporting, lysosomal 14 kDa, V1 subunit F  2.36 AJ130712 SIGLEC7sialic acid binding Ig-like lectin 7  2.36 NM_017905 TMCO3 transmembraneand coiled-coil domains 3  2.35 AB054985 CACNB1 calcium channel,voltage-dependent, beta 1 subunit  2.35 NM_005003 NDUFAB1 NADHdehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1, 8 kDa  2.35NM_001251 CD68 CD68 antigen  2.35 AA700869 PSCD2 Pleckstrin homology,Sec7 and coiled-coil domains 2 (cytohesin-2)  2.35 U94903 CD44 CD44antigen (homing function and Indian blood group system)  2.35 NM_003841TNFRSF10C tumor necrosis factor receptor superfamily, member 10c, decoywithout an intracellular domain  2.33 NM_004541 NDUFA1 NADHdehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5 kDa  2.33 BE567130KLRK1 Killer cell lectin-like receptor subfamily K, member 1  2.31NM_017460 CYP3A4 cytochrome P450, family 3, subfamily A, polypeptide 4 2.31 AI339536 DSC1 Desmocollin 1  2.31 NM_001783 CD79A CD79A antigen(immunoglobulin-associated alpha); CD79A antigen (immunoglobulin-associated alpha)  2.30 AA333161 VTI1A vesicle transport throughinteraction with t-SNAREs homolog 1A (yeast)  2.30 AW134823 CD6 CD6antigen; CD6 antigen  2.30 AL137537 ATP8B2 ATPase, Class I, type 8B,member 2  2.29 AI671983 SLC2A9 solute carrier family 2 (facilitatedglucose transporter), member 9  2.29 AA018187 C22orf3 chromosome 22 openreading frame 3  2.29 AL117415 ADAM33 ADAM metallopeptidase domain 33 2.29 NM_002588 PCDHGC3; protocadherin gamma subfamily C, 3; PCDHGB4;protocadherin gamma subfamily B, 4; PCDHGA8; protocadherin gammasubfamily A, 8; PCDHGA12; protocadherin gamma subfamily A, 12; PCDHGC5;protocadherin gamma subfamily C, 5; PCDHGC4; protocadherin gammasubfamily C, 4; PCDHGB7; protocadherin gamma subfamily B, 7; PCDHGB6;protocadherin gamma subfamily B, 6; PCDHGB5; protocadherin gammasubfamily B, 5; PCDHGB3; protocadherin gamma subfamily B, 3; PCDHGB2;protocadherin gamma subfamily B, 2; PCDHGB1; protocadherin gammasubfamily B, 1; PCDHGA11; protocadherin gamma subfamily A, 11; PCDHGA10;protocadherin gamma subfamily A, 10; PCDHGA9; protocadherin gammasubfamily A, 9; PCDHGA7; protocadherin gamma subfamily A, 7; PCDHGA6;protocadherin gamma subfamily A, 6; PCDHGA5; protocadherin gammasubfamily A, 5; PCDHGA4; protocadherin gamma subfamily A, 4; PCDHGA3;protocadherin gamma subfamily A, 3; PCDHGA2; protocadherin gammasubfamily A, 2; PCDHGA1 protocadherin gamma subfamily A, 1  2.29NM_020960 GPR107 G protein-coupled receptor 107  2.29 AK074635 GENX-3414Genethonin 1  2.29 BE138575 ITGB5 Integrin, beta 5  2.28 NM_003830SIGLEC5 sialic acid binding Ig-like lectin 5; sialic acid bindingIg-like lectin 5  2.28 NM_013319 UBIAD1 UbiA prenyltransferase domaincontaining 1  2.28 M63889 FGFR1 fibroblast growth factor receptor 1(fms-related tyrosine kinase 2, Pfeiffer syndrome)  2.27 H67156 MSCPSolute carrier family 25, member 37  2.27 BC006215 SMEK2 KIAA1387protein; KIAA1387 protein  2.27 AL109653 SLITRK2 SLIT and NTRK-likefamily, member 2  2.27 NM_007011 ABHD2 abhydrolase domain containing 2 2.26 AI767210 MGC11332 Hypothetical protein MGC11332  2.26 BF723605NRCAM Neuronal cell adhesion molecule  2.26 R08129 CDA08 T-cellimmunomodulatory protein  2.26 AF052059 SEL1L sel-1 suppressor oflin-12-like (C. elegans)  2.26 NM_005729 PPIF peptidylprolyl isomerase F(cyclophilin F)  2.25 BE858032 ARL2L1 ADP-ribosylation factor-like2-like 1  2.25 AI950390 C14orf118 Chromosome 14 open reading frame 118 2.24 NM_017767 SLC39A4 solute carrier family 39 (zinc transporter),member 4  2.24 AL110273 SPTAN1 Spectrin, alpha, non-erythrocytic 1(alpha-fodrin)  2.24 AI077660 CDA08 T-cell immunomodulatory protein 2.23 AA488687 SLC7A11 solute carrier family 7, (cationic amino acidtransporter, y + system) member 11  2.23 NM_000634 IL8RA interleukin 8receptor, alpha  2.22 AL390177 MGC34032 Solute carrier family 44, member5  2.21 NM_001531 MR1 major histocompatibility complex, class I-related 2.21 NM_003183 ADAM17 ADAM metallopeptidase domain 17 (tumor necrosisfactor, alpha, converting enzyme)  2.20 AC003999 SCAP2 src familyassociated phosphoprotein 2  2.20 BC014416 SLC33A1 solute carrier family33 (acetyl-CoA transporter), member 1  2.20 AF226731 ADORA3 adenosine A3receptor  2.19 AI608725 ICAM1 intercellular adhesion molecule 1 (CD54),human rhinovirus receptor  2.19 U41163 SLC6A8; solute carrier family 6(neurotransmitter FLJ43855 transporter, creatine), member 8; similar tosodium- and chloride-dependent creatine transporter  2.19 AU147799LRRC15 leucine rich repeat containing 15  2.18 AW337166 LOC255104Transmembrane and coiled-coil domains 4  2.18 NM_006505 PVR poliovirusreceptor  2.18 AI638420 CLIC4 chloride intracellular channel 4  2.18AI167482 SCUBE3 Signal peptide, CUB domain, EGF-like 3  2.18 AI739514HAS3 hyaluronan synthase 3  2.18 NM_005971 FXYD3 FXYD domain containingion transport regulator 3  2.17 AL022398 TRAF3IP3 TRAF3 interactingprotein 3  2.17 U90940 FCGR2C Fc fragment of IgG, low affinity IIc,receptor for (CD32)  2.16 B0023540 SORCS1 Sortilin-related VPS10 domaincontaining receptor 1  2.16 AV713913 OSTM1 osteopetrosis associatedtransmembrane protein 1  2.15 NM_024505 NOX5 NADPH oxidase, EF-handcalcium binding domain 5  2.15 BC006178 SEC22L3 SEC22 vesicletrafficking protein-like 3 (S. cerevisiae); SEC22 vesicle traffickingprotein- like 3 (S. cerevisiae)  2.15 BG151527 GRIK5 glutamate receptor,ionotropic, kainate 5  2.14 AW001754 NEGRI neuronal growth regulator 1 2.14 NM_013979 BNIP1 BCL2/adenovirus E1B 19 kDa interacting protein 1 2.14 NM_018643 TREM1 triggering receptor expressed on myeloid cells 1 2.12 NM_005284 GPR6 G protein-coupled receptor 6  2.11 AA454190 ZDHHC20zinc finger, DHHC-type containing 20  2.11 AB048796 TMPRSS13transmembrane protease, serine 13  2.11 AL044520 NYD-SP21 testesdevelopment-related NYD-SP21  2.11 BE463930 TMAP1 Matrix-remodellingassociated 7  2.10 NM_152264 SLC39A13 solute carrier family 39 (zinctransporter), member 13  2.08 AL530874 EPHB2 EPH receptor B2  2.07NM_018668 VPS33B vacuolar protein sorting 33B (yeast)  2.07 NM_024531GPR172A G protein-coupled receptor 172A  2.07 NM_023038 ADAM19 ADAMmetallopeptidase domain 19 (meltrin beta)  2.07 BC001281 TNFRSF10B tumornecrosis factor receptor superfamily, member 10b  2.07 AF217749 PCDHB9protocadherin beta 9  2.06 AB030077 FGFR2 fibroblast growth factorreceptor 2 (bacteria- expressed kinase, keratinocyte growth factorreceptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffersyndrome, Jackson-Weiss syndrome)  2.06 AL137432 SUSD1 sushi domaincontaining 1  2.05 NM_004518 KCNQ2 potassium voltage-gated channel,KQT-like subfamily, member 2  2.04 AI672363 VPS33B vacuolar proteinsorting 33B (yeast)  2.04 NM_006671 SLC1A7 solute carrier family 1(glutamate transporter), member 7  2.03 AA215519 DLGAP1 Discs, large(Drosophila) homolog-associated protein 1  2.02 NM_004648 PTPNS1 proteintyrosine phosphatase, non-receptor type substrate 1  2.02 NM_002564P2RY2 purinergic receptor P2Y, G-protein coupled, 2  2.01 BF511678SCUBE3 Signal peptide, CUB domain, EGF-like 3  2.01 BC013385 CLEC7AC-type lectin domain family 7, member A

CD47 Facilitates Engraftment, Inhibits Phagocytosis, and is More HighlyExpressed on AML LSC. It has long been recognized that the innate immunesystem, through natural killer (NK) effector cells, functions in theelimination of non-self and aberrant cells. NK cells eliminate targetcells recognized by a variety of NK cell-activating receptors that bindligands present on many normal cells; however, expression of self majorhistocompatibility complex (MHC) class I molecules can protect a cell bybinding to NK inhibitory receptors.

These inhibitory receptors often contain immunoreceptor tyrosine-basedinhibitory (ITIM) motifs that recruit and activate the SHP-1 and SHP-2tyrosine phosphatases, which in turn inhibit signal transduction fromthe activating receptors. Accumulating evidence indicates thatmonocyte-derived effector cells, such as macrophages and dendriticcells, are also involved in the elimination of non-self and aberrantcells, mediated by a number of activating receptors. These effectorcells also express the inhibitory receptor, signal regulatory proteinalpha (SIRPα), which contains an ITIM motif able to recruit and activatethe SHP-1 and SHP-2 phosphatases resulting in inhibition ofphagocytosis. Several studies have identified CD47 as the ligand forSIRPα. CD47 is a widely expressed transmembrane protein, originallyidentified as integrin associated protein (IAP) due to its physicalassociation with several integrins.

CD47 has been implicated in a number of processes including plateletactivation, cell motility and adhesion, and leukocyte adhesion,migration, and phagocytosis. The CD47-SIRPα interaction has beenimplicated in the inhibition of phagocytosis from a number of studies.First, CD47-deficient, but not wild type, mouse red blood cells (RBCs)were rapidly cleared from the bloodstream by splenic macrophages whentransfused into wild type mice, and this effect was dependent on theCD47-SIRPαinteraction. CD47-deficient, but not wild type, lymphocytesand bone marrow cells were also rapidly cleared upon transplantationinto congenic wild type recipients through macrophage and dendriticcell-mediated phagocytosis. Additional evidence suggested that theCD47-SIRPα interaction can inhibit phagocytosis stimulated by therecognition of IgG or complement opsonized cells. Thus, CD47 functionsas a critical regulator of macrophage and dendritic cell phagocytosis bybinding to SIRPα and delivering a dominant inhibitory signal.

We determined expression of CD47 on human AML LSC and normal HSC by flowcytometry. HSC (Lin−CD34+CD38−CD90+) from three samples of normal humanmobilized peripheral blood and AML LSC (Lin−CD34+CD38−CD90−) from sevensamples of human AML were analyzed for surface expression of CD47 (FIG.9). CD47 was expressed at low levels on the surface of normal HSC;however, on average, it was approximately 5-fold more highly expressedon AML LSC, as well as bulk leukemic blasts.

Anti-Human CD47 Monoclonal Antibody Stimulates Phagocytosis and InhibitsEngraftment of AML LSC. In order to test the model that CD47overexpression on AML LSC prevents phagocytosis of these cells throughits interaction with SIRPα on effector cells, we have utilized amonoclonal antibody directed against CD47 known to disrupt theCD47-SIRPα interaction. The hybridoma producing a mouse-anti-human CD47monoclonal antibody, termed B6H12, was obtained from ATCC and used toproduce purified antibody. First, we conducted in vitro phagocytosisassays. Primary human AML LSC were purified by FACS from two samples ofhuman AML, and then loaded with the fluorescent dye CFSE. These cellswere incubated with mouse bone marrow-derived macrophages and monitoredusing immunofluorescence microscopy (FIG. 10) and flow cytometry (FIG.11) to identify phagocytosed cells. In both cases, no phagocytosis wasobserved in the presence of an isotype control antibody; however,significant phagocytosis was detected with the addition of the anti-CD47antibody. Thus, blockage of human CD47 with a monoclonal antibody iscapable of stimulating the phagocytosis of these cells by mousemacrophages.

We next investigated the ability of the anti-CD47 antibody to inhibitAML LSC engraftment in vivo. Two primary human AML samples were eitheruntreated or coated with the anti-CD47 antibody prior to transplantationinto NOG newborn mice. 13 weeks later, the mice were sacrificed andanalyzed for human leukemia bone marrow engraftment by flow cytometry(FIG. 12). The control mice demonstrated leukemic engraftment while micetransplanted with the anti-CD47-coated cells showed little to noengraftment. These data indicate that blockade of human CD47 with amonoclonal antibody is able to inhibit AML LSC engraftment.

CD96 is a Human Acute Myeloid Leukemia Stem Cell-Specific Cell SurfaceMolecule. CD96, originally termed Tactile, was first identified as a Tcell surface molecule that is highly upregulated upon T cell activation.CD96 is expressed at low levels on resting T and NK cells and isstrongly upregulated upon stimulation in both cell types. It is notexpressed on other hematopoietic cells, and examination of itsexpression pattern showed that it is only otherwise present on someintestinal epithelia. The cytoplasmic domain of CD96 contains a putativeITIM motif, but it is not known if this functions in signaltransduction. CD96 promotes adhesion of NK cells to target cellsexpressing CD155, resulting in stimulation of cytotoxicity of activatedNK cells.

Preferential Cell Surface Expression of Molecules Identified from GeneExpression Analysis. Beyond CD47 and CD96, several of the moleculeslisted in FIG. 8B are known to be expressed on AML LSC, including:CD123, CD44, and CD33. The remaining molecules have not been previouslyreported or identified as preferentially expressed on human AML LSCcompared to their normal counterparts. We have examined cell surfaceexpression of two of these molecules by flow cytometry to determine ifthere is preferential expression on AML LSC compared to normal HSC.

CD99 is a surface glycoprotein with highest expression on T cells whereit may function in cellular adhesion. CD99 expression on HSC(Lin−CD34+CD38−CD90+) from three samples of normal human cord blood andAML LSC (Lin−CD34+CD38−CD90−) from seven samples of human AML wasdetermined by flow cytometry (FIG. 13). CD99 was expressed at low levelson the surface of normal HSC; however, on average, it is approximately5-fold more highly expressed on AML LSC. CD97 is normally expressed onmost mature hematopoietic cells and is upregulated on activatedlymphocytes where it may function in cellular migration and adhesion.Gene expression profiling indicates low to absent expression of CD97 inHSC and MPP, with approximately 10-fold higher expression in AML LSC.CD97 expression on normal cord blood HSC and AML LSC was examined byflow cytometry and found to be absent on HSC and high on 5 out of 7 AMLLSC samples (FIG. 14).

In order to evaluate the other candidate genes in FIG. 8B, we screenedthis list for those molecules not likely to be expressed on normal HSCbased on raw array expression values. Next, using published reports, weinvestigated the tissue expression pattern of these genes, in order toidentify those with very restricted patterns of expression for whichmonoclonal antibodies would have few targets besides the leukemia cells.Based on these methods, two promising genes were identified: ParathyroidHormone Receptor 2 and Hepatitis A Virus Cellular Receptor 2 (also knownas TIM-3: T cell immunoglobulin mucin 3). Parathyroid Hormone Receptor 2(PTHR2) is normally expressed in the pancreas and in some areas of thecentral nervous system. Its primary ligand is a peptide termedtuberoinfundibular peptide 39 (TIP39). Hepatitis A Virus CellularReceptor 2 (HAVCR2) is normally expressed on a subset of T lymphocytes.Its primary ligand is a molecule named Galectin-9.

Validation of additional sequences may utilize specific antibodies andtesting by flow cytometry, with comparison to normal HSC.

Example 3 Human and Mouse Leukaemias Upregulate CD47 to Evade MacrophageKilling

Tumour progression is characterized by several hallmarks, includinggrowth signal independence, inhibition of apoptosis, and evasion of theimmune system, among others. We show here that expression of CD47, aligand for the macrophage inhibitory signal regulatory protein alpha(SIRPα) receptor, is increased in human and mouse myeloid leukaemia andallows cells to evade phagocytosis and increase their tumorigenicpotential. CD47, also known as integrin associated protein (IAP), is animmunoglobulin-like transmembrane pentaspanin that is broadly expressedin mammalian tissues. We provide evidence that CD47 is upregulated inmouse and human myeloid leukaemia stem and progenitor cells, as well asleukemic blasts. Consistent with a biological role for CD47 in myeloidleukaemia development and maintenance, we demonstrate that ectopicover-expression of CD47 allows a myeloid leukaemia cell line to grow inmice that are T, B, and NK-cell deficient, whereas it is otherwisecleared rapidly when transplanted into these recipients. Theleukemogenic potential of CD47 is also shown to be dose-dependent, ashigher expressing clones have greater tumor forming potential than lowerexpressing clones. We also show that CD47 functions in promotingleukemogenesis by inhibiting phagocytosis of the leukemic cells bymacrophages.

CD47 is significantly upregulated in leukemic Fas^(lpr/lpr)×hMRP8bcl2transgenic bone marrow, and in leukemic hMRP8bcr/abl×hMRP8bcl2 mice.Transcripts for CD47 are increased in leukemic hMRP8bcr/abl×hMRP8bcl2bone marrow 3-4 fold by quantitative RT-PCR and 6-7 fold in c-Kitenriched leukemic marrow relative to healthy hMRP8bcl2+ bone marrow(FIG. 15e ). Leukemic spleen had an expansion of the granulocytemacrophage progenitor (GMP) population as well as c-Kit+ Sca-1+ Lin-stemand progenitor subsets relative to control mice, which were of the samegenotype as leukemic mice but failed to develop disease (FIGS. 15a-d ).Expression levels for CD47 protein were found to begin increasing inleukemic mice relative to control mice at the stage of the Flk2− CD34−c-Kit+Sca-1+Lin− long-term haematopoietic stem cell (LT-HSC) (FIG. 15f). This increased level of expression was maintained in GMP and Mac-1+blasts, but not megakaryocyte/erythroid restricted progenitors(MEP)(FIG. 15f ). The increase in CD47 between leukemic and normal cellswas between 3 to 20 fold. All mice that developed leukaemia that we haveexamined from hMRP8bcr/abl×hMRP8bcl2 primary (n=3) and secondarytransplanted mice (n=3), Fas^(lpr/lpr)×hMRP8bcl2 primary (n=14) andsecondary (n=19) mice, and hMRP8bcl2×hMRP8bcl2 primary (n=3) andsecondary (n=12) mice had increased CD47 expression. We have also foundincreased CD47 expression in mice that received p210bcr/ablretrovirally-transduced mouse bone marrow cells that developedleukaemia.

FACS-mediated analysis of human haematopoietic progenitor populationswas performed on blood and marrow derived from normal cord blood andmobilized peripheral blood (n=16) and myeloproliferative disorders(MPDs) including polycythemia vera (PV; n=16), myelofibrosis (MF; n=5),essential thrombocythemia (ET; n=7), chronic myelomonocytic leukaemia(CMML; n=11) and atypical chronic myeloid leukaemia (aCML; n=1) as wellas blast crisis phase chronic myeloid leukaemia (CML; n=19), chronicphase CML (n=7) and acute myelogenous leukaemia (AML; n=13). Thisanalysis demonstrated that granulocyte-macrophage progenitors (GMP)expanded in MPDs with myeloid skewed differentiation potential includingatypical CML, proliferative phase CMML and acute leukaemia includingblast crisis CML and AML (FIG. 16a ). AML HSC and progenitors uniformlyexhibited higher levels of CD47 expression compared with normal controls(FIG. 16b ); every sample from BC-CML and AML had elevated levels ofCD47. Moreover, progression from chronic phase CML to blast crisis wasassociated with a significant increase in CD47 expression (FIG. 16c ).Using the methods described in this study, we have found that human CD47protein expression in CML-BC increased 2.2 fold in CD90+ CD34+ CD38−Lin− cells relative to normal (p=6.3×10⁻⁵), 2.3 fold in CD90− CD34+CD38− Lin− cells relative to normal (p=4.3×10⁻⁵), and 2.4 fold in CD 34+CD38+ Lin− cells (p=7.6×10⁻⁶) (FIGS. 16b-16c ); however, using a neweroptimized staining protocol we have observed that CD47 is increasedapproximately 10 fold in AML and BC-CML compared to normal human HSCsand progenitors.

It was then asked whether forced expression of mouse CD47 on humanleukemic cells would confer a competitive advantage in forming tumoursin mice. MOLM-13 cells, which are derived from a patient with AML 5a,were transduced with Tet-MCS-IRES-GFP (Tet) or Tet-CD47-MCS-IRES-GFP(Tet-CD47) (FIG. 17a ), and stable integrants were propagated on thebasis of GFP expression. The cells were then transplanted intravenouslyin a competitive setting with untransduced MOLM-13 cells into T, B, andNK deficient recombination activating gene 2, common gamma chaindeficient (RAG2−/−, Gc−/−) mice. Only cells transduced with Tet-CD47were able to give rise to tumours in these mice, efficiently engraftingbone marrow, spleen and peripheral blood (FIGS. 17a-b ). The tumourswere also characterized by large tumour burden in the liver (FIGS. 17b,17g ), which is particularly significant because the liver is thought tohave the highest number of macrophages of any organ, with estimates thatKupffer cells may comprise 80% of the total tissue macrophagepopulation. These cells also make up 30% of the sinusoidal lining,thereby strategically placing them at sites of entry into the liver.Hence, significant engraftment there would have to disable a macrophagecytotoxic response. In addition to developing tumour nodules, theTet-CD47 MOLM-13 cells exhibited patterns of hepatic involvementtypically seen with human AML, with leukemic cells infiltrating theliver with a sinusoidal and perivenous pattern. (FIG. 17d ). Overall,Tet-CD47 MOLM-13 transplanted mice died more quickly than Tet MOLM-13transplanted mice, which had virtually no engraftment of leukemic cellsin hematopoietic tissues (FIG. 17c ). Tet-MOLM-13 mice still hadsignificant mortality, most likely due to localized growth at the siteof injection (retro-orbital sinus) with extension into the brain.

Since CD47 has been shown to be important for the migration ofhaematopoietic cells, and is known to modulate binding to extracellularmatrix proteins, either by direct interaction or via its effect onintegrins, one possibility for the lack of growth of Tet MOLM-13 cellsin mice was their inability to migrate to niches. To test thispossibility, Tet MOLM-13 or Tet-CD47 MOLM-13 cells were directlyinjected into the femoral cavity of immunodeficient mice. Tet-CD47MOLM-13 cells were able to engraft all bones and other haematopoietictissues of recipient mice, whereas Tet MOLM-13 cells had minimal, ifany, engraftment only at the site of injection (FIG. 17e ). Micetransplanted in this manner with Tet-CD47 MOLM-13 cells died atapproximately 50-60 days post-transplant (n=4), whereas mice thatreceived Tet MOLM-13 (n=5) cells remained alive for at least 75 dayswithout signs of disease at which point they were euthanized foranalysis. These results suggest a function other than or in addition tomigration or homing for CD47 in MOLM-13 engraftment.

Complete lack of CD47 has been shown to result in phagocytosis oftransplanted murine erythrocytes and leukocytes, via lack of interactionwith SIRPα on macrophages. Thus, we tested whether over-expression ofCD47 could prevent phagocytosis of live, unopsonized MOLM-13 cells. Weincubated Tet or Tet-CD47 MOLM-13 cells with bone marrow derivedmacrophages (BMDM) for 2-24 hours and assessed phagocytosis by countingthe number of ingested GFP+ cells under a microscope or by evaluatingthe frequency of GFP+ macrophages using a flow cytometer. Expression ofCD47 dramatically lowered macrophage clearance of these cells at alltime points tested, whereas Tet-MOLM-13 were quickly phagocytosed in amanner that increased overtime (FIGS. 18a-c ). We also injected MOLM-13cells into mice and analyzed hematopoietic organs 2 hours later forevidence of macrophage phagocytosis. Macrophages in bone marrow, spleen,and liver all had higher GFP+ fraction when injected with Tet MOLM-13cells as compared to CD47 expressing cells. This indicates that CD47over-expression can compensate for pro-phagocytic signals alreadypresent on leukemic cells, allowing them to survive when they wouldotherwise be cleared by macrophages.

Recent report indicates that lack of CD47 reactivity across speciesmight mediate xenorejections of transplanted cells. Furthermore, arecent study has demonstrated that human CD47 is unable to interact withSIRPα from C57Bl/6 mice, but is able to react with receptor fromnon-obese diabetic (NOD) mice, which are more permissive for human cellengraftment than C57Bl/6 mice. Furthermore, we have also observed thatHL-60 cells, a human promyelocytic cell line with higher levels of humanCD47 expression than MOLM-13, are able to engraft mice and causeleukaemia. Jurkat cells, a human T-lymphocyte cell line, are very highfor human CD47 and are phagocytosed by murine macrophages in vitro at amuch lower rate than MOLM-13. Thus, our data indicate that the abilityof cells to engraft mice in vivo or evade phagocytosis in vitro by mousemacrophages correlates with the level of human CD47 expression.

To model the tumorigenic effect of having high versus low CD47expression, we sorted clones of murine CD47 expressing MOLM-13 cellsinto high and low expressors. When adjusted for cell size, CD47 densityon the CD47^(lo) MOLM-13 cells was approximately equal to mouse bonemarrow cells, whereas CD47^(hi) MOLM-13 cells had approximately 9 foldhigher expression, an increase commensurate with the change seen in CD47expression on primary leukemic cells compared to their normalcounterparts (FIG. 19a ). When high or low expressing cells weretransplanted into recipients, only mice transplanted with highexpressing cells succumbed to disease by 75 days of age (FIG. 19c ).Furthermore, organomegaly was more pronounced in mice transplanted withhigh expressing cells (FIG. 19d ). Mice receiving CD47^(lo) MOLM-13cells still had notable liver masses. However, the masses wereinvariably 1-2 large nodes that were well-encapsulated and physicallysegregated from the liver parenchyma, in marked contrast to tumor massesfrom CD47hi MOLM-13 cells which consisted of hundreds of small massesscattered throughout the parenchyma. Thus, these large tumor massesconsist of cells which have found macrophage free-niches to grow inseparate from the main organ body. As expected, the infiltration ofMOLM-13 cells in bone marrow and spleen of recipient mice was muchhigher for mice transplanted with CD47^(hi) MOLM-13 cells as well (FIG.19e ). We also examined the level of CD47 expression in two mice thatreceived CD47^(lo) MOLM-13 cells but had significant marrow engraftment.In both cases, the persisting cells after 75 days had much higher levelsof CD47 than the original line (FIG. 19f ), indicating that a strongselection pressure exists in vivo for high levels of CD47 expression onleukemic cells. In total, these data indicate that CD47 expression levelis a significant factor in tumorigenic potential, and that in aheterogeneous population of leukemic cells, strong selection exists forthose clones with high CD47 expression.

We then asked if higher CD47 expression level would provide addedprotection against macrophage phagocytosis. We performed an in vitrophagocytosis assay with CD47^(hi) and CD47^(lo) MOLM-13 red fluorescentprotein (RFP) expressing cells. After incubation with macrophages, fargreater numbers of CD47^(lo) cells were phagocytosed as compared toCD47^(hi) cells (FIG. 19g ). If phagocytic indices are compared forcontrol MOLM-13 cells, bulk (un-sorted) CD47 MOLM-13 cells, CD47^(lo),and CD47^(hi) MOLM-13 cells, then a direct correlation between CD47expression level and ability to evade phagocytosis can be seen (FIG. 18a, FIG. 19f ). Furthermore, when CD47^(lo) RFP MOLM-13 cells andCD47^(hi) GFP MOLM-13 cells were co-incubated with macrophages in thesame wells, the low expressing cells were far more likely to bephagocytosed (FIGS. 19h, 19i ). Thus, in a mixed population of cellswith varying levels of CD47 expression, the low expressing cells aremore likely to be cleared by phagocytic clearance over time.

We also titrated CD47 expression using another method. Since CD47 isexpressed in MOLM-13 cells using a Tet-OFF system, we utilized theTet-inducible promoter element to control expression of CD47 in MOLM-13cells. Beginning two weeks after transplantation with CD47^(hi) MOLM-13cells, a cohort of mice was given doxycycline and followed for up to 75days post-transplant. During this time course, none of the mice givendoxycycline succumbed to disease or had large infiltration of MOLM-13cells in hematopoietic organs (FIGS. 19b-d ). At the doses ofdoxycycline used in this experiment, muCD47 expression in MOLM-13 cellswas reduced to levels below that of normal mouse bone marrow, butnotably not completely absent (FIG. 19b ). Thus, a sustained high levelof CD47 expression is required for robust MOLM-13 survival inhematopoietic organs.

Many examples of tumour clearance by T, B, and NK cells have beendescribed in the literature, indicating that a healthy immune system isessential for regulating nascent tumour growth. However, to date, fewexamples have been produced indicating that macrophage-mediatedphagocytosis can check tumour development. Collectively, our studiesreveal that ectopic expression of CD47 can enable otherwise immunogenictumour cells to grow rapidly in a T, B, and NK-cell deficient host.Furthermore, this is likely to reflect a mechanism used by human myeloidleukaemias to evade the host immune system since CD47 is consistentlyupregulated in murine and human myeloid leukaemias, including all formsof chronic and acute myeloid leukaemia tested thus far. Thus, it appearslikely that tumour cells are capable of being recognized as a target byactivated macrophages and cleared through phagocytosis. By upregulatingCD47, cancers are able to escape this form of innate immune tumoursurveillance.

This form of immune evasion is particularly important since thesecancers often occupy sites of high macrophage infiltration. CD47 wasfirst cloned as an ovarian tumour cell marker, indicating that it mayplay a role in preventing phagocytosis of other tissue cancers aswell¹⁸. Furthermore, solid tumours often metastasize to macrophage richtissues such as liver, lung, bone marrow, and lymph nodes, indicatingthat they must be able to escape macrophage-mediated killing in thosetissues. Finding methods to disrupt CD47-SIRPα interaction may thusprove broadly useful in developing novel therapies for cancer.Preventing CD47-SIRPα interaction could be doubly effective sinceantigens from phagocytosed tumour cells may be presented by macrophagesto activate an adaptive immune response, leading to further tumourdestruction.

Methods

Mice. hMRP8bcrabl, hMRP8bcl2, and Fas^(lpr/lpr) transgenic mice werecreated as previously described and crossed to obtain doubletransgenics. hMRP8bcl2 homozygotes were obtained by crossingheterozygote mice to each other. C57Bl/6 Ka mice from our colony wereused as a source of wild-type cells. For transplant experiments, cellswere transplanted into C57Bl/6 RAG2^(−/−) common gamma chain (Gc)^(−/−)mice given a radiation dose of 4 Gy using gamma rays from a cesiumirradiator (Phillips). Primary mouse leukemias were transplanted intoCD45.2 C57Bl6/Ka mice given a radiation dose of 9.5 Gy. Mice wereeuthanized when moribund.

Mouse tissues. Long bones were flushed with PBS supplemented with 2%fetal calf serum staining media (SM) Spleens and livers were dissociatedusing frosted glass slides in SM, then passed through a nylon mesh. Allsamples were treated with ACK lysis buffer to lyse erythrocytes prior tofurther analysis.

Quantitative RT-PCR Analysis. Bone marrow was obtained from leukemichMRP8bcr/abl×hMRP8bcl2 mice or hMRP8bcl2 control mice. Cells were c-Kitenriched using c-Kit microbeads and an autoMACS column (Miltenyi). RNAwas extracted using Trizol reagent (Invitrogen) and reversetranscription performed using SuperScriptII reverse polymerase(Invitrogen). cDNA corresponding to approximately 1000 cells was usedper PCR reaction. Quantitative PCR was performed with a SYBR green kiton an ABI Prism 7000 PCR (Applied Biosystems) machine at 50° C. for 2minutes, followed by 95° C. for 10 minutes and then 40 cycles of 95° C.for 15 minutes followed by 60° C. for 1 minute. Beta-actin and 18S RNAwere used as controls for cDNA quantity and results of CD47 expressionwere normalized. Sequences for 18S RNA forward and reverse primers wereTTGACGGAAGGGCACCACCAG and GCACCACCACCCACGGAATCG, respectively, forbeta-actin were TTCCTTCTTGGGTATGGAAT and GAGCAATGATCTTGATCCTC, and forCD47 were AGGCCAAGTCCAGAAGCATTC and AATCATTCTGCTGCTCGTTGC.

Human Bone Marrow and Peripheral Blood Samples. Normal bone marrowsamples were obtained with informed consent from 20-25 year old paiddonors who were hepatitis A, B, C and HIV negative by serology (AllCells). Blood and marrow cells were donated by patients with chronicmyelomonocytic leukemia (CMML), chronic myeloid leukemia (CML), andacute myelogenous leukemia (AML) and were obtained with informedconsent, from previously untreated patients.

Cell lines. MOLM-13 cells were obtained from DSMZ. HL-60 and Jurkatcells were obtained from ATCC. Cells were maintained in Iscove'smodified Dulbecco's media (IMDM) plus 10% fetal bovine serum (FBS)(Hyclone). To fractionate MOLM-13 cells into those with high and lowCD47 expression, Tet-CD47 MOLM-13 cells were stained with anti-mouseCD47 Alexa-680 antibody (mIAP301). The highest and lowest 5% of mouseCD47 expressing cells was sorted on a BD FACSAria and re-grown inIMDM+10% FCS for 2 weeks. The cells were sorted for three more rounds ofselection following the same protocol to obtain the high and lowexpressing cells used in this study. To obtain red fluorescent protein(RFP) constructs, the mCherry RFP DNA was cloned into Lentilox 3.7(pLL3.7) empty vector. Lentivirus obtained from this construct was thenused to infect cell lines.

Cell staining and flow cytometry. Staining for mouse stem and progenitorcells was performed using the following monoclonal antibodies: Mac-1,Gr-1, CD3, CD4, CD8, B220, and Ter119 conjugated to Cy5-PE (eBioscience)were used in the lineage cocktail, c-Kit PE-Cy7 (eBioscience), Sca-1Alexa680 (e13-161-7, produced in our lab), CD34 FITC (eBioscience),CD16/32 (FcGRII/III) APC (Pharmingen), and CD135 (Flk-2) PE(eBioscience) were used as previously described to stain mouse stem andprogenitor subsets. Mouse CD47 antibody (clone mIAP301) was assessedusing biotinylated antibody produced in our lab. Cells were then stainedwith streptavidin conjugated Quantum Dot 605 (Chemicon). Samples wereanalyzed using a FACSAria (Beckton Dickinson).

For human samples, mononuclear fractions were extracted following Ficolldensity centrifugation according to standard methods and analyzed freshor subsequent to rapid thawing of samples previously frozen in 90% FCSand 10% DMSO in liquid nitrogen. In some cases, CD34+ cells wereenriched from mononuclear fractions with the aid of immunomagnetic beads(CD34+ Progenitor Isolation Kit, Miltenyi Biotec, Bergisch-Gladbach,Germany). Prior to FACS analysis and sorting, myeloid progenitors werestained with lineage marker specific phycoerythrin (PE)-Cy5-conjugatedantibodies including CD2 RPA-2.10; CD11b, ICRF44; CD20, 2H7; CD56, B159;GPA, GA-R2 (Becton Dickinson—PharMingen, San Diego), CD3, S4.1; CD4,S3.5; CD7, CD7-6B7; CD8, 3B5; CD10, 5-1B4, CD14, TUK4; CD19, SJ25-C1(Caltag, South San Francisco, Calif.) and APC-conjugated anti-CD34,HPCA-2 (Becton Dickinson-PharMingen), biotinylated anti-CD38, HIT2(Caltag) in addition to PE-conjugated anti-IL-3Rα, 9F5 (BectonDickinson-ParMingen) and FITC-conjugated anti-CD45RA, MEM56 (Caltag)followed by staining with Streptavidin—Texas Red to visualize CD38-BIOstained cells.

Following staining, cells were analyzed using a modified FACS Vantage(Becton Dickinson Immunocytometry Systems, Mountain View, Calif.)equipped with a 599 nm dye laser and a 488 nm argon laser or a FACSAria.Hematopoietic stem cells (HSC) were identified as CD34+ CD38+ CD90+ andlineage negative. Anti-human CD47 FITC (clone B6H12, Pharmingen) wasused to assess CD47 expression in all human samples. Fold change forCD47 expression was determined by dividing the average mean fluorescenceintensity of CD47 for all the samples of CML-BC, CML-CP, or AML by theaverage mean fluorescence intensity of normal cells for a given cellpopulation. Common myeloid progenitors (CMP) were identified based onCD34+ CD38+ IL-3Rα+ CD45RA− lin− staining and their progeny includinggranulocyte/macrophage progenitors (GMP) were CD34+CD38+IL-3Rα+ CD45RA+Lin− while megakaryocyte/erythrocyte progenitors (MEP) were identifiedbased on CD34+ CD38+ IL-3Rα− CD45RA− Lin− staining.

To determine the density of mouse or human CD47, cells were stained withsaturating amounts of anti-CD47 antibody and analyzed on a FACSAria.Since forward scatter is directly proportional to cell diameter, anddensity is equal to expression level per unit of surface area we usedFloJo software to calculate geometric mean fluorescent intensity of theCD47 channel and divided by the geometric mean of the forward scattervalue squared (FSC²) to obtain an approximation for density of CD47expression on the membrane.

Engraftment of MOLM-13 cells was assessed by using anti-human CD45PE-Cy7 (Pharmingen), anti-mouse CD45.2 APC (clone AL1-4A2), andanti-mouse CD47 Alexa-680 (mIAP301). All samples were resuspended inpropidium iodide containing buffer before analysis to exclude deadcells. FACS data was analyzed using FloJo software (Treestar).

Lentiviral preparation and transduction.pRRL.sin-18.PPT.Tet07.IRES.GFP.pre, CMV, VSV, and tet trans-activator(tTA) plasmids were obtained from Luigi Naldini. The full length murinecDNA for CD47 form 2 was provided by Eric Brown (UCSF). The CD47 cDNAconstruct was ligated into the BamHI/NheI site of Tet-MCS-IRES-GFP.Plasmid DNA was transfected into 293T cells using standard protocols.The supernatant was harvested and concentrated using a Beckman LM-8centrifuge (Beckman). Cells were transduced with Tet orTet-CD47-MCS-IRES-GFP and tTA lentivirus for 48 hours. GFP+ cells weresorted to purity and grown for several generations to ensure stabilityof the transgenes.

Injections. Cells were injected intravenously into the retro-orbitalsinuses of recipient mice or via the tail vein as noted. Forintra-femoral injections, cells were injected into the femoral cavity ofanesthetized mice in a volume of 20 μl using a 27-gauge needle. Anisofluorane gas chamber was used to anesthetize mice when necessary.

MOLM-13 cell engraftment. Animals were euthanized when moribund and bonemarrow, spleen, and liver harvested. Peripheral blood was obtained bytail bleed of the animals 1 hour prior to euthanization. Engraftment ofMOLM-13 cells in marrow, spleen, and peripheral blood was determined asdescribed above. Tumor burden in the liver was determined by calculatingthe area of each visible tumor nodule using the formula ((length inmm+width in mm)/2)*π. Area of each nodule was then added together perliver.

Doxycycline administration. Doxycycline hydrochloride (Sigma) was addedto drinking water at a final concentration of 1 mg/mL. Drinking waterwas replaced every 4 days and protected from light. In addition, micereceived a 10 μg bolus of doxycline by i.p. injection once a week.

Bone marrow derived macrophages (BMDM). Femurs and tibias were harvestedfrom C57Bl/6 Ka mice and the marrow was flushed and placed into asterile suspension of PBS. The bone marrow suspension was grown in IMDMplus 10% FBS with 10 ng/mL of recombinant murine macrophage colonystimulating factor (MCSF, Peprotech) for 7-10 days.

In vitro phagocytosis assays. BMDM were harvested by incubation intrypsin/EDTA (Gibco) for 5 minutes and gentle scraping. Macrophages wereplated at 5×10⁴ cells per well in a 24-well tissue culture plate(Falcon). After 24 hours, media was replaced with serum-free IMDM. Afteran additional 2 hours, 2.5×10⁵ Tet or Tet-CD47 MOLM-13 cells were addedto the macrophage containing wells and incubated at 37° C. for theindicated times. After co-incubation, wells were washed thoroughly withIMDM 3 times and examined under an Eclipse T5100 (Nikon) using anenhanced green fluorescent protein (GFP) or Texas Red filter set(Nikon). The number of GFP+ or RFP+ cells within macrophages was countedand phagocytic index was calculated using the formula: phagocyticindex=number of ingested cells/(number of macrophages/100). At least 200macrophages were counted per well. For flow cytometry analysis ofphagocytosis macrophages were harvested after incubation with MOLM-13cells using trypsin/EDTA and gentle scraping. Cells were stained withanti-Mac-1 PE antibody and analyzed on a BD FACSAria. Fluorescent andbrightfield images were taken separately using an Eclipse T5100 (Nikon),a super high pressure mercury lamp (Nikon), an endow green fluorescentprotein (eGFP) bandpass filter (Nikon) a Texas Red bandpass filter(Nikon), and a RT Slider (Spot Diagnostics) camera. Images were mergedwith Photoshop software (Adobe).

For in vivo assays, marrow from leg long bones, spleen, and liver wereharvested 2 hours after injecting target cells into RAG2−/−,Gc−/− mice.They were prepared into single cell suspensions in PBS plus 2% FCS.Cells were labeled with anti-human CD45 Cy7-PE and anti-mouse F4/80biotin (eBiosciences). Secondary stain was performed withStreptavidin-APC (eBiosciences). Cells that were human CD45-, F4/80+were considered to be macrophages, and GFP+ cells in this fraction wasassessed.

What is claimed is:
 1. A method of treating a human subject for apre-leukemic condition, the method comprising: administering to a humansubject in need thereof an antibody that disrupts the binding of CD47with SIRPα in combination with an antibody that specifically binds toHAVCR2, at a dose that achieves a depletion in pre-leukemic cells byincreasing phagocytosis of pre-leukemic cells.
 2. The method of claim 1,wherein the antibody that disrupts the binding of CD47 with SIRPαspecifically binds to CD47.
 3. The method of claim 1, wherein theantibody that disrupts the binding of CD47 with SIRPα is a humanized orchimeric monoclonal antibody.
 4. The method of claim 1, wherein thepreleukemic condition is a myelodysplastic syndrome.
 5. The method ofclaim 4, wherein the myelodysplastic syndrome is selected from chronicmyelogenous leukemia, polycythemia vera, essential thrombocytosis,agnogenic myelofibrosis and myeloid metaplasia.
 6. The method of claim1, wherein the human subject is further treated with a chemotherapeuticdrug.
 7. The method of claim 1, wherein the antibody that specificallybinds to HAVCR2 is a humanized or chimeric monoclonal antibody.